[Heartlander]

Is there a case where the penicillinase enzyme was observed to evolve in a population where it did not already exist?

[allmendream]

Evolution of penicillin resistance, yes - by mutations of beta lactamase (penicillinase) enzymes such that penicillin is a better substrate, and by mutations that cause over-expression of those enzymes.

The basic structure itself capable of breaking down Penicillin like compounds is already present - but in penicillin susceptible bacteria the antibiotic is not a good substrate for the enzyme and it is not produced in sufficient quantity.