Question

Which of the following is not a step in producing penicillin -G-amidase?

(a) Cleavage by Hind III

(b) Replacement of asparagine residue with serine present in the native enzyme

(c) Cleaved fragments cloned into a cosmid vector

(d) Penicillin-G-amidase gene transferred on to pBR322 plasmids

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This interesting question is from Future Prospects topic in chapter Enzyme Technology Future Prospects of Enzyme Technology

Answer 1

The correct option is (b) Replacement of asparagine residue with serine present in the native enzyme

Best explanation: Replacement of asparagine residue with serine present in the native enzyme is a step involved in site directed mutagenesis and not in production of penicillin –G-amidases. The steps involved are as follows:

• Cleavage of DNA by Hind III at relatively rare sites containing the 5′-AAGCTT-3′.

• The cleaved DNA is cloned into a cosmid vector and then returned to E. coli.

• Followed by selection of colonies containing the active gene by their inhibition of a 6-amino-penicillanic acid-sensitive organism.

• Such colonies are isolated and the penicillin-G-amidase gene is transferred on to pBR322 plasmids and re-cloned back into E. coli.

• The engineered cells produce penicillin-G-amidase constitutively and in considerably higher quantities than parental strain.