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Cell products > L10 Biocatalysis > Flashcards

L10 Biocatalysis Flashcards

1
Q

What disease is L-Dopa used for?

A

Parkinson’s disese

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2
Q

What is the physiology of disease of PD?

A

Degeneration of substantia nigra - decreased production of dopamine

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3
Q

Does dopamine cross the BBB?

A

No

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4
Q

Does L_Dopa cross the BBB?

A

Yes

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5
Q

How is L-Dopa made?

A

Microbial manufacture by using tyrosine phenol lyase

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6
Q

What are process parameters that affect TPL prod.?

A

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

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7
Q

What is the medium composition for TPL?

A

Carbon/nitrogen
NaCl
Inducer (tyrosine)

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8
Q

What company uses microorganisms to make L-DOpa?

A

Ajinomoto Chemical company

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9
Q

What mo does ACC use to make L-Dopa?

A

Whole cells of Erwinia herbicola

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10
Q

What are problems with using tyrosine as an inducer?

A

Contaminates final product

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11
Q

How did the company overcome tyrosine contamination?

A

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

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12
Q

What was a problem with E.Coli?

A

Production not as good

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13
Q

What is tpl promoter activated by?

A

TyrR in presence of tyr

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14
Q

How is mutant E. herbicola made?

A

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

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15
Q

What does tyrR mutagenesis allow for?

A

production of TPL without tyrosine addition

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16
Q

What are the 3 aa changes in TyrR?

A

V67A
Y72C
E201G

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17
Q

What is acrylamide used for?

A

paints
Adhesives
paper

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18
Q

When is polyacrylamide used for?

A

Water treatment - flocculation
Gel electrophoresis

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19
Q

What is acrylamide made from?

A

Acrylonitrile - toxic

20
Q

What do some m/o use acrylonitrile as?

A

carbon-energy sources

21
Q

What does nitrile hydratase do/

A

Hydrolysis of nitrile to amide

22
Q

What does amidase do?

A

Converts amide to ammonia + carboxylic acid

23
Q

What does chemical manufacture of acrylamide require?

A

Copper catalyst

24
Q

What is the microbial process of acrylamide?

A

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

25
Q

What was one of the OG strains used for acrylamide?

A

Pseudomonas chloraphis B23

26
Q

What are 3 characteristics of Pseudomonas chloraphis B23?

A

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

27
Q

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

A

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

28
Q

How was the biocatalyst optimised?

A

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

29
Q

What was the problem with using sucrose?

A

Mucilage

30
Q

How was mucilage overcome?

A

Turned to random mutagenesis

31
Q

What biocatalysis for acrylamide improved productivity?

A

Am324

32
Q

What is a 3rd generation biocatalyst?

A

Rhodococcus rhodochrous j1 - contains cobalt atom

33
Q

What is RrJ1 better?

A

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

34
Q

What was added as an inducer to RrJ1?

A

Urea

35
Q

What are 4 gene cloning & overexpression advantages?

A

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

36
Q

What are 4 disadvantages of gene cloning & overexpression?

A

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

37
Q

What do disadvantages were solved by synthetic genes?

A

Codon usage
Introns

38
Q

What are 4 steps involved in overexpression?

A

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

39
Q

What genes are ligated to a vector?

A

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

40
Q

How do you make cells competent?

A

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

41
Q

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

A

Ampilician - only cells containing plasmid will grow

42
Q

How do you induce expression?

A

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

43
Q

What host of e.coli most commonly used?

A

BL21

44
Q

What mutations does BL21 have?

A

Mutations that prevent protein & plasmid degradation

45
Q

What promoter does BL21 have?

A

T7 - bacterial chromosome, very strong

46
Q

What is T7 controlled by?

A

lac promoter

47
Q

What switches on T7 RNA polymerase expression?

A

IPTG

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