Supplementary information-

A strategic approach for direct recovery and stabilization of Fusarium sp. ICT SAC1

cutinase from solid state fermented broth by carrier free cross-linked enzyme

aggregates

Sandeep A. Chaudhari1, and Rekha S. Singhal1 *

1Food Engineering and Technology Department, Institute of Chemical Technology, Matunga,

Mumbai-400019, India

*Corresponding authors:

Prof. Rekha S. Singhal

rsinghal7@reddiffmail.com

rs.singhal@ictmumbai.edu.in

Total page: 06

Total figures: 07

1

1. FTIR

The FTIR data were recorded with 2.0 cm-1 resolution, within analytical range of 4000 -

400 cm-1, at room temperature (28 ± 2 °C) and total 100 spectral scan were collected. Three

replica spectra were collected in order to assess its reproducibility. Bands were identified by

comparison with earlier published assignments.

Fig S1. pH optima and stability of free and cut-CLEAs under different pH conditions.

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.00

20

40

60

80

100

120Free Cutinase cut-CLEAs

pH

Rel

ativ

e A

ctiv

ity (%

)

Fig S2. Temperature optima of free and cut-CLEAs at different temperatures.

20 25 30 35 40 45 50 550

20

40

60

80

100

120

Temperature (°C)

Rel

ativ

e A

ctiv

ity (%

)

2

Fig S3. Thermal studies of free cutinase [F] and cut-CLEA[C]; A) thermal stability; B) TG

and DTG curves (––) TG; (- - - -) DTG.

Free CutinaseCut-CLEA

Temperature ( � C)

Wei

ght l

oss (

%)

Der

ivat

ive w

eigh

t los

s (%

)

3

Fig S4. Correlation of log P, dielectric constant and dipole moment of solvents with

cutinase activity.

Fig. S4A

5.6 4.57 3.5 3.15 0.63 0.58 0.34 0.32 -1.3 -0.23

0.28 -0.3 -0.76

0102030405060708090

100

Free Cutinase cut-CLEA

log P values of solvents tested

Rel

ativ

e A

ctiv

ity (%

)

Fig. S4B

46.68 32

.724

.621

.0120

.3319

.9217

.93 17.8 8.1 3.4 2.2

5 2 1.90

102030405060708090

100

Free cutinase cut-CLEA

Dielctric constant (ε) values of solvents tested

Rel

ativ

e A

ctiv

ity (%

)

4

Fig. S4C

46.7 2.88 1.76 1.7 1.69 1.68 1.66 1.66 1.64 1.6 0.45 0.07 00

102030405060708090

100

Free Cutinase cut-CLEA

Dipole moment of solvent

Rel

ativ

e A

ctiv

ity (%

)

Fig S5. Lineweaver-Burk plot (1/S vs. 1/V) of free cutinase and cut-CLEA.

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.20

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

f(x) = 0.0581869745256428 x − 0.0143893884253717R² = 0.946389481357465

f(x) = 0.0821690478871904 x − 0.0186016820007986R² = 0.984385561872146

f(x) = 0.046575576187062 x − 0.011646347399791R² = 0.944382513013829

Free cutinase Linear (Free cutinase)BSA cut-CLEA Linear (BSA cut-CLEA)

1/S (mM)

1/V

(µm

ol/m

in)

5

Fig. S6. Recyclability of cut-CLEA

0 1 2 3 4 5 6 7 8 9 10 1110

20

30

40

50

60

70

80

90

100

Number of reuse cycles

Rel

ativ

e ac

tivity

(%)

Fig. S7. Physical appearance of wet and dried cut-CLEA.

6

Wet cut-CLEA Dry cut-CLEA