Abstract:
:Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned, expressed in pET-28-E. coli expression system as a heterodimeric enzyme with molecular weights of 45 and 20 kDa for large and small subunit, respectively. It was purified by nickel affinity chromatography with hydrolytic and transpeptidase activity of 1.82 U/mg and 4.35 U/mg, respectively. Sequence analysis revealed that GGTBP was most closely related to Bacillus licheniformis GGT and had all the catalytic residues and nucleophiles for autoprocessing recognized from E. coli. It was optimally active at pH 8 and 60°C. It exhibited pH stability from pH 6-9 and high thermostability with t(1/2) of 15 min at 70°C. It had K(m), V(max) of 0.045 mM, 4.35 μmol/mg/min, respectively. Decoupling of autoprocessing by co-expressing large and small subunit in pET-Duet1-E. coli expression system yielded active enzyme with transpeptidase activity of 5.31 U/mg. Though N-terminal truncations of rGGTBP upto 95 aa did not affect autoprocessing of GGT however activity was lost with truncation beyond 63 aa.
journal_name
Enzyme Microb Technoljournal_title
Enzyme and microbial technologyauthors
Murty NA,Tiwary E,Sharma R,Nair N,Gupta Rdoi
10.1016/j.enzmictec.2011.08.005subject
Has Abstractpub_date
2012-03-10 00:00:00pages
159-64issue
3eissn
0141-0229issn
1879-0909pii
S0141-0229(11)00187-6journal_volume
50pub_type
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