L10 Biocatalysis

Question 1

What disease is L-Dopa used for?

Answer 1

Parkinson’s disese

Question 2

What is the physiology of disease of PD?

Answer 2

Degeneration of substantia nigra - decreased production of dopamine

Question 3

Does dopamine cross the BBB?

Answer 3

No

Question 4

Does L_Dopa cross the BBB?

Answer 4

Yes

Question 5

How is L-Dopa made?

Answer 5

Microbial manufacture by using tyrosine phenol lyase

Question 6

What are process parameters that affect TPL prod.?

Answer 6

pH (7.5)
Inoculum age & volume (6% v/v of 4 day old culture)

Question 7

What is the medium composition for TPL?

Answer 7

Carbon/nitrogen
NaCl
Inducer (tyrosine)

Question 8

What company uses microorganisms to make L-DOpa?

Answer 8

Ajinomoto Chemical company

Question 9

What mo does ACC use to make L-Dopa?

Answer 9

Whole cells of Erwinia herbicola

Question 10

What are problems with using tyrosine as an inducer?

Answer 10

Contaminates final product

Question 11

How did the company overcome tyrosine contamination?

Answer 11

Used gene tpl expressed in recombinant E.coli with strong promoter instead

Question 12

What was a problem with E.Coli?

Answer 12

Production not as good

Question 13

What is tpl promoter activated by?

Answer 13

TyrR in presence of tyr

Question 14

How is mutant E. herbicola made?

Answer 14

Knocked out gene tyrR
Transformed with a plasmid containing mutant tyrR
3 aa substitutions - enhanced activation

Question 15

What does tyrR mutagenesis allow for?

Answer 15

production of TPL without tyrosine addition

Question 16

What are the 3 aa changes in TyrR?

Answer 16

V67A
Y72C
E201G

Question 17

What is acrylamide used for?

Answer 17

paints
Adhesives
paper

Question 18

When is polyacrylamide used for?

Answer 18

Water treatment - flocculation
Gel electrophoresis

Question 19

What is acrylamide made from?

Answer 19

Acrylonitrile - toxic

Question 20

What do some m/o use acrylonitrile as?

Answer 20

carbon-energy sources

Question 21

What does nitrile hydratase do/

Answer 21

Hydrolysis of nitrile to amide

Question 22

What does amidase do?

Answer 22

Converts amide to ammonia + carboxylic acid

Question 23

What does chemical manufacture of acrylamide require?

Answer 23

Copper catalyst

Question 24

What is the microbial process of acrylamide?

Answer 24

Exposing water & acrylonitrile to immobilized m/o -> hydration -> separation -> decoloring -> product

Question 25

What was one of the OG strains used for acrylamide?

Answer 25

Pseudomonas chloraphis B23

Question 26

What are 3 characteristics of Pseudomonas chloraphis B23?

Answer 26

Does not grow on acrylonitrile Grows on isobutyronitrile Resting cells convert 99% acrylonitrile

Question 27

What are 3 steps to making acrylonitrile using m/o?

Answer 27

Culture cells on isobutynitrile on carbon source Harvest cells Resuspend cells in buffer of acrylonitrile

Question 28

How was the biocatalyst optimised?

Answer 28

Alter medium (sucrose) Inducer now methyacrylamide Got 40% of soluble protein

Question 29

What was the problem with using sucrose?

Answer 29

Mucilage

Question 30

How was mucilage overcome?

Answer 30

Turned to random mutagenesis

Question 31

What biocatalysis for acrylamide improved productivity?

Answer 31

Am324

Question 32

What is a 3rd generation biocatalyst?

Answer 32

Rhodococcus rhodochrous j1 - contains cobalt atom

Question 33

What is RrJ1 better?

Answer 33

More efficient than B23 More resistant to heat, substrate & product

Question 34

What was added as an inducer to RrJ1?

Answer 34

Urea

Question 35

What are 4 gene cloning & overexpression advantages?

Answer 35

More protein Eliminate unwanted enzyme additives Mutagenesis/molecular evolution Purification of enzyme is easier

Question 36

What are 4 disadvantages of gene cloning & overexpression?

Answer 36

Inclusion bodies - mis-folding Codon usage - varies between species Introns - not present in prokaryotes Posttranslational modifications - glycosylation

Question 37

What do disadvantages were solved by synthetic genes?

Answer 37

Codon usage Introns

Question 38

What are 4 steps involved in overexpression?

Answer 38

Obtain gene of interest Insert into expression vector Transform host Induce expression

Question 39

What genes are ligated to a vector?

Answer 39

trc promoter - RNA polymerase binds strongly lacO - operator region where repressor protein binds lacI - codes for repressor protein

Question 40

How do you make cells competent?

Answer 40

Electroporation - makes cell envelope permeable Ice-cold CaCl2 - heat shock - cells take up plasmids

Question 41

What is used for plate transformants on antibiotic - containing plate?

Answer 41

Ampilician - only cells containing plasmid will grow

Question 42

How do you induce expression?

Answer 42

Culture transformant - no inducer present allows cells to grow without trying to express foreign gene Add inducer (IPTG) - expression of foreign gene Purify protein

Question 43

What host of e.coli most commonly used?

Answer 43

BL21

Question 44

What mutations does BL21 have?

Answer 44

Mutations that prevent protein & plasmid degradation

Question 45

What promoter does BL21 have?

Answer 45

T7 - bacterial chromosome, very strong

Question 46

What is T7 controlled by?

Answer 46

lac promoter

Question 47

What switches on T7 RNA polymerase expression?

Answer 47

IPTG