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4.13. Cloning and expression of cyanamide hydratase (cah) marker
gene
4.13.1. Site-directed rnutagenesis in calr gene
Sequencing data of mutated crrh indict~ted the single hilse suhst~tution, G to A
ot nucleotide position 81 or T to C at nucleotide position 81 of ccrl~ gene (Fig. 23). The
one base substitution does not change [he amino acid it encodes since both AAG
(wild) and AAA (cah MutK mutant) encode lysine and the codons. CTT (wild) and
CTC (cuh MutL mutant) encode leucine.
4.13.2. Cloning and overexpression of wild and mutant type cah genes in E. coli
Recombinant plasmids, pQ-cah (wild-type), pQ-rahMutK and pQ-i.ohMutL
(mutant- type) were constructed through cloning of amplified mutated rah genes into
pQE-60 (Fig. 24). IPTG induction of E. coli carrying wild-type pQ-ruh as well as
mutants, pQ-cahMutK and pQ-cahMutL showed overexpression of rah as his-tag
fusion protein.
4.13.3. Purification of enzyme and cyanamide hydratase assay
Cell-free crude enzyme preparations were purified using ~ i ' ' NTA agarow
column. Crude enzyme preparations of pQ-cah, pQ-cahMutK and pQ-cuhMutL
showed Cah enzyme activity of 1090, 1060 and 1000 Ulmg protein respectively.
Purified enzyme exhibited 5-fold higher activity than the crude enzyme preparation
(pQ-cah.
5200;
pU-cahMutK, 5200 and pQ-cahMutL 5100 Ulrng protein).
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