Berry Lectures 4-6

Question 1

what activity does lipase possess

Answer 1

esterase activity

Question 2

briefly describe how was lipase screened for stereoselectivity

Answer 2

select and grow colonies and assay with R and S stereoselectivity. overlapping wells are non-specific but any which are only yellow with R are R specific and vice versa

Question 3

what substrate was used to screen for lipase stereoselectivity

Answer 3

fatty acid ester with paranitrophenol which is yellow when cleaved

Question 4

what is the E value of WT lipase

Answer 4

1.1

Question 5

what was the first step in the experiment to creat a stereospecific lipase

Answer 5

error prone PCR with an error rate of 2codons/gene to generate 1 aa change per gene and therefor 5400 possible mutants. this was repeated a number of times

Question 6

what were the mutations in lipase after 5 rounds of error prone PCR (selecting for best mutant after each round and using this as template for next round)

Answer 6

S149G, S155L, V47G, F259L, L110R

Question 7

after 6 rounds of error prone PCR, what was the e value of lipase

Answer 7

15

Question 8

after error prone PCR, what was the next step in the experiment to create a stereoselective lipase

Answer 8

they identified hotspots for mutations and used site directed mutagenesis to test each amino acid at that residue and determine which was best. when they changed 155L for F they found the E value increased considerably

Question 9

why is the route taken through the landscape important

Answer 9

because you may get interplay between beneficial and detrimental mutations e.g. a single mutation of S155F significantly increased E value, as did the subsequent addition of S164G and V55G but the addition of S149G to the single mutant reduces activity so backcrossing is important to prom removal of this mutation

Question 10

what are the 2 possible outcomes of the reaction of DHAP and G3P

Answer 10

TBP or FBP dependent on which enzyme is used