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- Title
Refolding of Misfolded Inclusion Bodies of Recombinant α-Amylase: Characterization of Cobalt Activated Thermostable α-Amylase from Geobacillus SBS-4S.
- Authors
Mansoor, Sabah; Tayyab, Muhammad; Jawad, Amna; Munir, Bushra; Firyal, Sehrish; Awan, Ali Raza; Rashid, Naeem; Muhammad Wasim
- 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.
- Subjects
ALPHA-amylase; COBALT; ESCHERICHIA coli; CARBOXYPEPTIDASES; NUCLEOTIDE sequencing
- Publication
Pakistan Journal of Zoology, 2018, Vol 50, Issue 3, p1147
- ISSN
0030-9923
- Publication type
Article
- DOI
10.17582/journal.pjz/2018.50.3.1147.1155