Laboratory Evolution of CotA Laccase for

Increased Half-life at pH 4 Sunandan Dhar1, Silu Sheng2, and Edgardo T. Farinas2

1 Department of Biotechnology, Heritage Institute of Technology, Kolkata, India 700107 2 Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark NJ 07102

Engineer proteins to optimize

properties

DNA sequence is randomly

mutated and expressed (1-2

mutations per gene)

Screening of library of

mutants for desired parameters

Iterative process

Protein surface display is a technique used to screen protein li-

braries.

Recently, bacterial endospores have been demonstrated to be an

effective microbial display tool [1]:

No protein folding problems

Preimmobilized

Stable in extreme conditions

Easy to engineer

CotA protein on spore coat of

B. subtilis:

Laccase enzyme activity [2]

Oxidizes variety of sub-

strates (copper dependent)

Maximum catalytic activity in

pH 4, but highly unstable

After screening 3000 clones, a

triple mutant (E498G, E29V,

L343S) was found. However,

only E498G is responsible for

increased half-life. This mutant

was further characterized.

Wild-type B. subtilis and mutant cells are grown on LB plate, then

transferred to Schaeffer plate. Sporulation is induced by incuba-

tion is 2SG media (nutrient starvation) for 72 hours at 37⁰C.

Purification of spores

Washing with salt solution & water

Centrifuge at 17000 R.P.M.

Dark brown spores obtained

Study of Kinetics

Activity: oxidation of ABTS

substrate

Acid inactivation: spores

suspended in pH 4 buffer

Vmax = 13.88 μM/ min/ OD unit

Km = 63.54 μM

Vmax = 32.77 μM/ min/ OD unit

Km = 473.65 μM

Objective: Increase stability in pH 4 using Directed Evolution

Wild-type: t½ = 21 min

E498G : t½ = 869 min 41 fold Improvement of Stability

Kinetic parameters of mutant slightly hindered

Significant improvement of stability in acidic conditions

Industrial & biotechnological applications

Further research to improve catalytic activity

0

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14

0 200 400 600 800 1000 1200

Activi

ty (

μM

/m

in /O

D u

nit)

ABTS Concentration (μM)

Wild-type Kinetics

0

5

10

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20

25

30

35

0 1000 2000 3000 4000 5000 6000 7000 8000 9000

Activity (μ

M /m

in /O

D u

nit)

ABTS Concentration (μM)

E498G Kinetics

Directed Evolution

Procedure

Results

Conclusion

References:

[1] Gupta, Nirupama, et al. “Directed evolution of CotA laccase for increased substrate specificity using

Bacillus subtilis spores.” Protein Engineering, Design & Selection 23.8 (2010): pp 679-682.

[2] Martin, Ligia, et al. “Molecular and Biochemical Characterization of a Highly Stable Bacterial Laccase

That Occurs as a Structural Component of the Bacillus subtilis Endospore Coat.” The Journal of

Biological Chemistry 227.21 (2002): pp18849-18859.

Acknowledgements: National Science Foundation Closed circle: t = 0 min Open circle: t = 120 min

0%

20%

40%

60%

80%

100%

120%

0 20 40 60 80 100 120 140 160 180 200

Rela

tive A

ctivity (%

)

Time (minutes)

Acid Inactivation

Wild-type

E498G