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 oryzae NCIM 1212 using bran, varying chemical parameters under solid state

M.V.V.Chandana Lakshmi* and Perumallapallis Jyothi

Department of Chemical Engineering, AUCE(A),Andhra University, Visakhapatnam-03, Andhra Pradesh, .(Corresponding author)

*Corresponding author

A B S T R A C T

Glucoamylase is a well recognized amylolytic used in food industry, which is generally produced by, Aspergillus niger, Aspergillus wentii, Aspergillus terreus K e y w o r d s and . Its production was optimized under solid state fermentation in potato dextrose agar medium. Different substrates like wheat bran, 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: 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- that breaks the units from the non can hydrolyze into , 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 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). 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 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 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 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 , 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 ; 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 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. The process parameters was to evaluate the released glucose was measured with 3, 5- effect of an individual parameter at a time dinitrosalicyclic acid (DNS) reagent using and to incorporate it at the standard level. glucose as a standard (Miller, 1959). Glucoamylase activity unit (U) was Effect of chemical parameters expressed as the amount of enzyme releasing one mole of glucose equivalent Effect of carbon source per minute per ml.

Influence of various carbon supplements One unit of glucoamylase was defined as on enzyme production was carried out by the amount of enzyme required to release adding various carbon supplements 1 mol of glucose/min under the assay namely glucose, fructose, maltose, starch conditions and activity was expressed as and a 1% (w/w) to fermentation U/mL. The activity was also expressed as medium. U/mg biomass, and U/mg proteins. Similar assays for glucoamylase have been used Effect of organic and nitrogen source by other researchers (Mahajan et al. 1983)

Influence of various organic nitrogen Standard curve (DNS Method for supplements on enzyme production was Glucose) studied by adding various supplements namely urea, soya bean meal, peptone, Dinitro salicylate reagent yeast extract, extract sodium nitrate, ammonium sulphate, ammonium chloride Dissolve 1.6gm of NaoH in 20ml of and potassium nitrate at 1% (w/w) to distilled water, then dissolve 1g of 3,5- fermentation medium Dinitro salicylate acid in above NaoH solution. Dissolve 20gm of sodium Extraction of crude enzyme potassium tartarate in 50ml of H2O in a separate beaker and mix with dinitro To the fermented dough 50 mM citrate

73 Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 70-76 salicylic acid solution and finally make up on enzyme production was studied by to 100ml with distilled water. adding various supplements namely glucose, fructose, maltose, starch and Standard solution of glucose (1mg/ml) sucrose a 1% (w/w) to fermentation media. Of the five different carbon supplements Pipette out different volumes of glucose used as enrichment, fructose was a good (0.2-1ml) into different test tubes and carbon supplements which gave maximum make the volume to 1ml with water. Add glucoamylase activity 42.32 U/ml (Figure 1ml of dintrosalicylate acid in each test 2). Fructose was the best carbon tube and keep in a boiling water bath for supplements, as it provides the much 10min. Cool and add 10ml of distilled required carbon in substrate. water in each test tube and measure the orange red colour developed at 520nm in a Zambare et al (2010) reported maximum photoelectric calorimeter. production of amylase when fructose was the carbon supplement. Calculation Effect of organic nitrogen supplements Unit (U/ml) = (mg of glucose on glucoamylase production liberated)(1000)/(molecular weight of glucose)(Volume of supernatant used in Influence of various organic nitrogen calorimetric determination) supplements on enzyme production was Volume of supernatant used in studied by adding various supplements calorimetric determination=1ml namely urea, soya bean meal, peptone, yeast extract and malt extract at 1% (w/w) Results and discussion to fermentation media. Of the five In the present study agro-residues and different organic nitrogen supplements microorganisms were screened for their used as enrichment, peptone was a good potential to serve as substrate and fungal nitrogen supplements which gave supplements for glucoamylase production maximum glucoamylase production in SSF. The process parameters for the 45.21U/ml.(figure 3). Peptone was the best production of glucoamylase by Aspergillus organic nitrogen supplements as it oryzae NCIM 1212 strain grown on wheat provides the much required nitrogen in bran substrate were done under SSF. For substrate. enzyme production process of chemical parameters like the basal medium was Imran et al (2011) reported that, peptone supplemented with different nutrients in enhanced the glucoamylase production the form of carbon supplements, organic and inorganic nitrogen supplements. The Effect of inorganic nitrogen results are presented and discussed in view supplements on glucoamylase of existing literature. production Chemical parameters Influence of various inorganic nitrogen supplements on enzyme production was Effect of carbon supplements on studied by adding various supplements glucoamylase production namely sodium nitrate, ammonium sulphate, ammonium chloride, potassium Influence of various carbon supplements nitrate at 1% (w/w) to fermentation media. 74

Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 70-76

Fig.1 Glucose Standard Curve

Fig.2 Effect of carbon supplements on glucoamylase

Fig.3 Effect of Organic nitrogen supplements on glucoamylase

75 Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 70-76

Of the five different organic nitrogen References supplements used as enrichment, ammonium sulphate was a good inorganic Box, G. E., W.G. Hunter and Hunter, J.S.2005. supplement which gave maximum Statistics for Experimenters: Design, glucoamylase activity 32.25 U/ml (Figure Innovation, and Discovery , 2nd Edition, 4) but when compared with organic Wiley. supplements it is lower therefore inorganic Box, G. E. P., W.G. Hunter and Hunter, S. nitrogen supplements has no effect on J.1978. Statistics for Experimenters , John glucoamylase activity. Wiley & Sons, Inc, New York, 1978. Choi, H., S.R. Al-Abed, D.D. Dionysiou, E. Glucoamylases are the most widely used Stathatos and Lianoss, P.2010. TiO2 Based enzymes in industries. In the present work, Advanced Oxidation Nanotechnologies for four different substrates and Water Purification and Reuse , microorganisms were screened. It was Sustainability Sci. Engineer. 2: 229-254. found that, maximum activity of Jun Wang, W., Z. Zhaohong Zhang , Y. Rui Xu glucoamylase 36.31U/ml was found using and Xiangdong, Z.2007. Treatment of nano-sized rutile phase TiO2 powder under wheat bran with A.oryzae NCIM1212. ultrasonic irradiation in hydrogen peroxide Laboratory scale optimization of some of solution and investigation of its fermentation conditions for glucoamylase sonocatalytic activity , ultrasonic production by A.oryzae NCIM 1212 in sonochemistry. SSF was carried out. The chemical Manmohan Lal Kamboj, 2000. Studies on the parameters like carbon and nitrogen degradation of industrial waste water using supplements. The nutritional heterogeneous photocatalysis , Department supplementation study was carried out of biotechnology and environmental using various carbons, nitrogen (organic sciences, Thapar University, Patiala, pp.1- and inorganic supplements) to enrich the 67. production medium to enhance the enzyme Naresh, N., Mahamuni and Aniruddha B. yield. The optimized conditions are: Pandit. 2006. Effect of additives on Carbon source: Addition of Fructose as ultrasonic degradation of phenol , carbon source showed high glucoamylase Ultrasonic Sonochem.13:165 174. activity of 42.32 U/ml compared to other Rajeshwar, K., J.G. Ibanez and Swain, carbon sources like glucose, maltose, G.M.1994. Electrochemistry and the sucrose, and soluble starch. Nitrogen Environment , J. Appl. Electrochem. source: Addition of peptone as organic 24:1077. nitrogen supplement showed high Sandra Contreras Iglesias.2002. Degradation glucoamylase activity of 45.21U/ml than and biodegradability enhancement of other nitrogen sources like yeast extract, nitrobenzene and 2, 4-dichlorophenol by malt extract, urea and soya bean meal. means of advanced oxidation processes Addition of Ammonium sulphate as based on ozone , Barcelona, pp.1-22. Suneetha Parameswarappa, Chandrakant inorganic nitrogen supplement showed Karigar and Manjunath Nagenahalli. 2008. reduced glucoamylase activity. As well as Degradation of ethylbenzene by free and optimization parameters carried out in this immobilized , Biodegrad.19:137 144. study proved to be fruitful in enhancing US EPA.,1996. Priority pollutants , Code programs for enzymes of biotechnological of Federal regulations, Title 40, Chapter importance. 1, Part 423, Appendix A. Environmental Protection Agency, Washington, DC.

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