Production and Optimization of Glucoamylase from Aspergillus Oryzae NCIM 1212 Using Wheat Bran, Varying Chemical Parameters Under Solid State Fermentation
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Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 70-76 ISSN: 2319-7706 Volume 3 Number 5 (2014) pp. 70-76 http://www.ijcmas.com Original Research Article Production and optimization of Glucoamylase from Aspergillus oryzae NCIM 1212 using wheat bran, varying chemical parameters under solid state fermentation M.V.V.Chandana Lakshmi* and Perumallapallis Jyothi Department of Chemical Engineering, AUCE(A),Andhra University, Visakhapatnam-03, Andhra Pradesh, India.(Corresponding author) *Corresponding author A B S T R A C T Glucoamylase is a well recognized amylolytic enzyme used in food industry, which is generally produced by, Aspergillus niger, Aspergillus wentii, Aspergillus terreus K e y w o r d s and Aspergillus oryzae. Its production was optimized under solid state fermentation in potato dextrose agar medium. Different substrates like wheat bran, rice bran and Glucoamylase, coconut oil cake which were purchased from the local market are checked for Aspergillus optimum production. During screening it was found that wheat bran using species, Solid Aspergillus oryzae was more productive than any other source. Optimized state production was obtained by varying the chemical parameters such as carbon fermentation, supplements, Organic and inorganic nitrogen supplements. The following chemical observations were made: Fructose as a carbon supplement gave activity 42.32 parameters U/ml. Peptone acted as a good nitrogen supplement resulting in activity of 45.21 U/ml. Ammonium sulphates as a good inorganic nitrogen supplement, gave an activity of 32.25 U/ml. Introduction Glucoamylase (E.C. 3.2.1.3) is an enzyme glucoamylase (EC 3.2.1.3). a- Amylase that breaks the glucose units from the non can hydrolyze starch into maltose, glucose reducing sides of amylase chain, glycogen and maltotriose by cleaving the 1, 4-a-D- and amyl pectin involving in the glucosidic linkages between adjacent hydrolysis of (1-4) faster than (1-6), glucose units in the linear amylase chain. (1-3) linkages and produce producing D Glucoamylase hydrolyses single glucose glucose in successive manner (Joshi VK, units from the non-reducing ends of Pandey A et al (1999) Vasudeo Zambare amylase and amyl pectin in a stepwise 2010). The amylase family has two major manner, and produce glucose as the sole classes, namely amylase (EC 3.2.1.1) and end-product from starch and related 70 Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 70-76 polymers. Unlike a-amylase, most al (2013). and Aspergillus oryzae in glucoamylases are also able to hydrolyze enzyme industry (Shruti Puri et al (2013) the 1, 6-a-linkages at the branching points Biesbeke R et al (2005). Amylases are of amyl pectin, although at a lower rate universally distributed throughout the than 1, 4-linkages (Fogarty, W.M, (1983), animal, plant and microbial kingdoms. Antranikian.G, (1992), RiteshParbat et al Spectrum of application of amylases has (2011). widened in many sectors such as clinical, medicinal and analytical chemistry. Glucoamylases are industrially important Current developments in bio-technology hydrolytic enzymes of biotechnological are yielding new applications for enzymes. significance and are currently used in food In the last decades, there has been an and pharmaceutical industries. (Joshi VK, increasing trend towards the utilization of Pandey A et al (1999), Vasudeo Zambare the solid state fermentation (SSF) to (2010), James JA and Lee BH (1992). produce several enzymes from micro- organisms (Shruti Puri et al (2013) Sodhi Glucoamylases mainly used in the H K et al 2005). The food, beverage and production of glucose syrup, high fructose agro industries produce large quantities of corn syrup, and alcohol (James JA and Lee residues that pose serious problems of BH, 1992).Glucoamylases are produced disposal, in spite of them being sources of by various microorganisms, including biomass and nutrients. These substrates bacteria; fungi and yeasts, but almost are used for production of valuable exclusively in filamentous fungi and far compounds such as enzymes and various less in bacteria and yeast (RiteshParbat et secondary metabolites (Shruti Puri et al al 2011). These enzymes have found wide (2013) Soccol C R et al (2003). Agro- applications in the processed food industrial residues are generally industry, fermentation industry, and textile considered the best substrates for SSF and paper industries (Selvakumar, et al processes and use of SSF for the (1999) Pandey A et al (1999) Tengerdy production of enzymes is no exception to RP, 1998) that (Shruti Puri et al (2013) Ellaiah P et al (2002). Use of suitable low cost Traditionally, glucoamylase has been fermentation medium for production of produced by SmF and used in a one-way alpha amylase using agricultural by- process in solution. In recent years, products has been reported (Haq et al however, the solid state fermentation 2003). Wheat bran, paddy husk, rice (SSF) processes have been increasingly processing waste and other starch applied for the production of this enzyme. containing wastes have gained importance SSF holds tremendous potential for the as supports for growth during enzyme production of enzymes (Pandey A et al production (Anto et al 2006). 1999). It can be of special interest in those processes where the crude fermented Sources and forms of gluco amylases: product may be used directly as enzyme source (Tengerdy RP, 1998). Glucoamylase s may be derived from a wide variety of plants, animals and The exclusive production of microorganisms, though most glucoamylases is achieved by Aspergillus glucoamylase s occur in fungi. The niger (Wang X J et al (2006) Shruti Puri et enzymes used commercially originate 71 Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 70-76 from strains of either Aspergillus Niger or pressure for sterilization. Then tubes were Rhizopus sp. where they are used for the laid in the slanting position for the conversion of malto-oligosaccharides into solidification of the medium (Rashid et al glucose (Fogarty et al (1983) and Pandey 1997). The slants were inoculated with et al 1995). These enzymes are generally spores of desire organisms and finally regarded as safe (GRAS) by the Food and placed in incubator at 25 - 30°C for 5-6 Drug Administration (FDA). The days. Fully matured slants were stored at properties of glucoamylase have been 4°C and renewed once a month. reviewed comprehensively elsewhere. Since the discovery of two forms of 1. Aspergillus niger NCIM 616 glucoamylase from black koji mold in the 2. Aspergillus oryzae NCIM 1212 1950 s, many reports have appeared on the 3. Aspergillus terreus MTCC 1782 multiplicity of glucoamylase. The various 4. Aspergillus wentii MTCC 1901 forms of glucoamylases are thought to be the result of several mechanisms: mRNA Maintenance of culture modifications, limited proteolysis, variation in carbohydrate content or the The cultures were maintained on PDA presence of several structural genes slants and incubated for 5 days at 28 °C. (Pretorius et al 1991). The organisms were sub cultured every month. Subtrates: Commonly used carbon sources are dextrin; fructose, glucose, Inoculum preparation lactose; maltose and starch are very expensive for commercial production of A piece of culture from 5 days old slant these enzymes. various agricultural by was used to inoculate in seed flask products like wheat bran (WB),groundnut containing 5g substrate with 100% oil cake (GOC), rice husk (RH),coconut moisture and incubated for 5 days at 30°C. oil cake (COC) sugarcane bagasee (SB) After incubation, fermented dough was potato residue (PR) rice brain (RB),green mixed aseptically followed by addition of gram bran (GGB),black gram bran (BGG) 50 ml of saline containing 0.1% Tween- and maize bran (MB)etc are abuntely used 80. After 20 min the mixture was filtered off through sterile glass wool to get spores. Spore count was determined by Materials and Methods serial dilution and spread plating method Microorganisms and Maintenance Enzyme production in Solid state fermentation (SSF) The organisms used in the present study were procrued from the National The static experiments were conducted in Collection of Industrial Microorganisms 250 ml Erlenmeyer flasks containing 5 g (NCIM), Pune and The Microbial Type of substrates and then moistened with 5ml Culture Collection and Gene Bank of water and sterilized at 121 °C for 30 (MTCC), Chandigarh. The cultures were min. The fermentation process was started maintained on potato dextrose agar slants. by adding 1 ml of spore suspension (5 x Sporulation medium was poured into 107 spores/ml) as prepared above. The culture test tubes and autoclaved for 15 whole content was mixed thoroughly and minutes at 121°C temperature and 20 psi then incubated at 30°C for 5 days. 72 Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 70-76 Screening of substrates buffer (pH 5) (1:10) was added and homogenized for 2 hours with a constant The substrates used in this study namely stirring at room temperature. This groundnut oil cake, rice bran, wheat bran suspension was filtered through Whatman and coconut oil cake were purchased from filter paper number 1 and the filtrate was local market in Visakhapatnam. These again centrifuged at 6000 rpm for 15 min. substrates were dried in an oven at 105°C This solid-free supernatant was used as to a constant weight and then used to study enzyme source for assaying glucoamylase their effect on the glucoamylase activity. production. The best solid substrate achieved by this step was fixed for Assay For Glucoamylase subsequent experiments. (Ritesh Parba and Barkha Singhal, 2011) Optimization process Glucoamylase activity was determined by taking an appropriate amount of the Fermentation process is governed by a enzyme followed by the addition of 1 % large number of chemical factors. The soluble starch solution in 50 mM citrate protocol adopted for the optimization of buffer (pH=5.5) at 50°C for 20 min.