Production and Optimization of Glucoamylases from Plants and Aspergillusniger for Starch Hydrolysis in a Batch Bioreactor
Production and Optimization of Glucoamylases from Plants and Aspergillusniger for Starch Hydrolysis in a Batch Bioreactor.
ABSTRACT
Glucoamylases were produced from both plants and microorganisms and were optimized for starch hydrolysis in batch bioreactor. Amylase activity was monitored in germinating guinea corn seeds for seven days. Highest amylase activity was observed on days 3 and 7. A study of the amylopectin content of millet, guinea corn, cassava, corn and tigernut starch showed that tiger nut had the highest amylopectin content while cassava starch had the lowest.
Moist amylopectin frommillet, guinea corn, cassava, corn and tiger nut starch were exposed on the shelf to triger microbial growth. Luxurial growths were noticed on amylopectin from guinea corn, tigernut and cassava starch. Pure isolates were obtained by subculturing and identified as Aspergillus niger. A 14-day fermentation study to determine the optimal production time using the organism and amylopectin from guinea corn, tigernut and cassava starch was carried out.
The fermentation studies showed a two-peak profile for each amylopectin used.The first on day 3 or 4, while the second peak on day 11 and 12, respectively. Large scale production of glucoamylase was carried out on these days of highest enzyme production. Glucoamylase activities from both germinating guinea corn seeds and Aspergillus niger were enhanced by calcium (Ca2+), zinc (Zn2+), cobolt (Co2+), iron (Fe2+) and manganese (Mn2+) ionbut Lead ion (Pb2+) completely inactivated the enzymes.
The Michaelismenten constant (Km) and the maximum velocity (Vmax)obtained from Lineweaver-Burk plot of initial velocity data at different substrate concentrations showed high affinity of the glucomylases for their substrates. The optimal pH and temperature of glucoamylases from both germinating seeds and Aspergillus niger were in the range of 4.5-8.5 and 45-60 ˚C, respectively.
INTRODUCTION
The huge demand for starch in industries for the production of high glucose syrups and ethanol has led to stiff competition with dietary starch. There is the need to discover other sources of starch for industrial purposes in other to spare dietary starch. This is complicated by the problems of completely hydrolysing starch due to the difficulty of obtaining the appropriate enzymes to hydrolyse its multiple branching especially theα- 1, 6 glucosidic bonds.
The study of plant and microbial glucoamylases is important from both basic and applied perspectives. Amylase is a major enzyme in the industry (Souzaand Magalhaes, 2010). It hydrolyses the starch molecules into glucose units (Raimi et al., 2012). Cereal grains synthesize multiple forms of α-amylase during germination to supply soluble carbohydrates for the developing seedling.
Heterogeneity of starch- degrading enzymes in germinating seeds enhances the conversion of insoluble granules to soluble starch and dextrins (Donn et al., 1991). These multiple forms of amylase suggest that each isoform may have a particular metabolic function. The individual forms act cooperatively to degrade starch during germination. The exclusive production of amylases has also been reported in Aspergillus niger, A. oryzae, A flavus and A. terreus(Zambare, 2010; Koc and Metin, 2010; Puriet al., 2013; Lawal et al., 2014).
The most well known amylolytic enzymes are α-amylase (EC 3.2.1.1), β-amylase (EC3.2.1.2) and glucoamylase (EC 3.2.1.3) (Sivaramakrishnan et al., 2006; Janecek, 2009). α-Amylase digests starch by randomly breaking the glycosidic bonds between glucose molecules. The product is therefore a mixture of maltose and dextrins. β-amylase digests starch by cleaving every second bond starting from one end, producing maltose.
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CSN Team.
