Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymesReport as inadecuate

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Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R and D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases EC, GH57 from an endogenous Cohnella sp., Coh00831 KP335161; 1998 bp and Coh01133 KP335160: 3678 bp were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 DE3. The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose G2, maltotriose G3 and maltotetraose G4. The enzymes hydrolyzed pullulan to maltotriose G3 only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.

Author: Fatemeh Zebardast Roodi, Saeed Aminzadeh , Naser Farrokhi, AliAsghar Karkhane, Kamahldin Haghbeen



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