Purification and characterization of Thermostable and Calcium independant - Amylase from Streptomyces griseus TBG19NRA1
Balakrishnan Divya1, Sugathan Shiburaj1 and Nediyaparambu Sukumaran Pradeep1,
1Division of Microbiology, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram-695 562 (India) 1KSCSTE- Malabar Botanical Garden and Institute of Plant Science, Kozhikode-673014 (India) Email: drdivyaullas2006@gmail.com, drsashiburaj@gmail.com,drnspradeep@gmail.com Mob: +91 8129718203
ABSTRACT
The alpha amylase is one of the most important industrial enzyme which has wide range of commercial interest. The amylolytic enzymes active at high temperatures (90oC) and calcium independent would benefit starch-processing industries. A mesophilic isolate of Streptomyces griseus TBG19NRA1 produces extracellular thermo stable α-amylase. The strain showed maximum α-amylase activity at the end of 48 h of incubation at pH 7. The enzyme was purified and molecular weight of 60 kDa was obtained. The purified α-amylase reported maximum relative activity at 80oC without the addition of CaCl2 and revealed the enzyme is thermostable in nature. The amylase activity was strongly stimulated by Fe3+ and Mg2+ (5 mM). The chemical inhibitors and denaturants like DTT, BME and EDTA slightly stimulated enzymatic activity. The analysis of kinetic showed that the enzyme resulted Km of 1.6 mg/mL and V max of 28 mg/mL/min at pH 7. The Peptide mass fingerprinting using LC-MS analysis and subsequent Uniprot database search confirmed the enzyme as α-amylase. The present studies focus on the optimization of production conditions and purification of thermostable α-amylase from S. griseus TBG19NRA1.The purified alpha amylase were thermostable at 80°C and calcium independent in nature. The enzyme purifies were suitable in liquefaction of starch in high temperature and in starch based industrial applications.