You have identified an enzyme from a thermophilic bacterium that lives in hot sp

Question

You have identified an enzyme from a thermophilic bacterium that lives in hot springs which converts glucose into a compound that effectively treats male pattern baldness. When you express your enzyme in E. Coli for mass production of your compound, you do not recover any compound from protein expressing bacterial culture grown in standard laboratory conditions at 37C.

1) Hoping to create a variant with higher activity in E. Coli, you perform error prone PCR to introduce random mutations into the gene. You isolate a more active variant, and determine that it has a very similar molecular eright but a much lower extinction coefficient at 280 nm. Explain why this variant might be a more active E. Coli.

Explanation / Answer

Answer 1) We know that the proteins which are functional at high temperature are very well tightly packed so that the high temperature must not denature their structure or break their stabilizing bonds. but such tight packing/arrangement in a protein might interfere with its catalysis ability or enzymatic activity. after error-prone PCR, one mutated protein becomes more active variant but its molecular mass remains same. though it has much lower extinction coefficient at 280nm. it somehow suggests that the synthesized concentration of this enzyme is low but there might have occurred some changes in its ligand-binding pocket (active site) due to alteration of some amino acid in that sequence. it led to more efficient binding and enhanced catalysis of glucose into the desired compound.

In short, error-prone PCR introduced some changes in its amino acid sequence such that the active site of the protein is more accessible to the glucose and enhanced rapid catalysis is observed.

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