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Refolding of Misfolded Inclusion Bodies of Recombinant α-Amylase: Characterization of Cobalt Activated Thermostable α-Amylase from Geobacillus SBS-4S

Refolding of Misfolded Inclusion Bodies of Recombinant α-Amylase: Characterization of Cobalt Activated Thermostable α-Amylase from Geobacillus SBS-4S

Sabah Mansoor1, Muhammad Tayyab1,*, Amna Jawad1, Bushra Munir2, Sehrish Firyal1, Ali Raza Awan1, Naeem Rashid3 and Muhammad Wasim1

1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Abdul Qadir Jillani Road, Lahore
2Institute of Industrial Biotechnology, Government College University, Lahore
3School of Biological Sciences, University of The Punjab, Quaid-e-Azam Campus, Lahore

*         Corresponding author: muhammad.tayyab@uvas.edu.pk

 

ABSTRACT

The present study deals with the production, refolding and characterization of recombinant α-amylase (AMYSBS) from Geobacillus SBS-4S. AMYSBS exhibited a highest identity of 99.78% with Geobacillus thermoleovorance GTA. E.coli BL21-CodonPlus (DE3) cells were used as host for expression studies of AMYSBS. Recombinant AMYSBS produced as inclusion bodies was transmitted to soluble active form by denaturing the insoluble protein using 8M urea followed by refolding through gradual dialysis. The refolded enzyme exhibited optimum activity at 55 °C between pH 8-9. The effect of metal ions on the activity of AMYSBS showed that Co2+ remarkably enhanced the enzyme activity and 500µM was recorded as optimal Co2+ concentration for the maximal activity of AMYSBS. Presence of ionic (SDS) and nonionic (Tween-20, TritonX-100) detergents showed an enhancing effect on the activity of AMYSBS. Stability studies of AMYSBS exhibited that enzyme was quiet stable at 55 °C. Kinetic studies demonstrated the Km and Vmax values of 6.67mg/ml and 2500µmol min-1 mg-1, respectively when starch was utilized as substrate. To best of our knowledge this is the highest activity among the reported recombinant amylases from genus Geobacillus. Laboratory scale production of reducing sugars from cloth-starch makes AMYSBS a suitable candidate to be used in Textile industry.

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Pakistan Journal of Zoology

October

Pakistan J. Zool., Vol. 56, Iss. 5, pp. 2001-2500

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