Glucose dehydrogenase (GDH) is an enzyme that catalyzes the chemical reaction: D

Question

Glucose dehydrogenase (GDH) is an enzyme that catalyzes the chemical reaction:

D-glucose + NADP+ -> D-glucose-1,5,lactone +NADPH

you are asked to improve the organic solvent tolerance (up to 50% DMSO ) of GDH

a) based on your knowledge, which method do you prefer to enigeer this specific enzyme, rational design OR directed evolution and explain why?

b) now you decide to use directed evolution, and error-prone PCR is used to creat a GDH mutant library. If the mutation rate is controlled to be 1 nucelotide per 1 kb DNA, what is expected library size(# of different protein mutants create) on amino acid level?

c) What are the library selection and screening methods in directed evolution experiments? To identify the beneficial GDH mutants, will you use a selection or screening method based on the information provided in the question? Explain how you will perform it (Hint: You may need to design your selection or screening method based on the reaction provided).

d) After several rounds of engineering, an enzyme mutant was obtained to show much higher DMSO tolerance. Would you expect this mutant to catalyze the reaction in hexane at a higher rate than that of the wild-type enzyme? Assume this enzyme is functional in buffer with certain hexane percentage.

Explanation / Answer

Solution:

a) Rational method have been successfully use to a glucose dehydrogenase enzyme because to thermal stability, EDTA tolerance and substrate selectivity

Rational protein design implies the use of knowledge to design or improve the characteristic of bio molecule

b) If the mutation rate is controlled to be 1 nucleotide per equivalent 1nt/ kb DNA then expected library size is /kb

Error prone PCR protocols are effective at changing the DNA sequence

In contrast single nucleotide insertion and more commonly single nucleotide insertion and deletion, are produced but at a much lower rate

c) Library selection in directed evolution experiment is mutant library of glucose dehydrogenase genes

Ultra high throughput screening method is used in directed evolution experiment

Related Questions

Navigate

• Browse (All)
• Browse G
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.