Supplementary Tables and Figures
Table 1 Comparative biochemical properties of wild and recombinant TALip54
Properties
Lip54
rTALip54
Protein type
wild
recombinant
pH optima
9.0
8.0
Temperature optima
40 °C
50 °C
Substrate specificity
p-Nitrophenyl myristate
p-Nitrophenyl laurate
Enantioselectivity
Solvent based enantio
inversion
Kumar et al. (2009)
Solvent based enantio
inversion
Current research paper
References
Supplementary Figure Legends
1a: Schematic representation of overlapping PCR to amplify TAlipC
1b: Amplification of TALipC gene from genomic DNA of Trichosporon asahii MSR54
Lane 1:- molecular marker
Lane 2:- Truncated gene amplified from cDNA
Lane 3:- Full gene with introns amplified from gDNA
Lane 4:- Full functional gene TAlipC amplified by overlapping PCR
1c: Native PAGE analysis of deglycosylate enzyme
Deglycosylation was performed according to manufacturer’s instruction given in deglycosylation
kit from New England Biolabs (Lot No. 0041403). Native ladders are shown in lane one, second
and third lane corresponds to glycosylated and de-glycosylated TALipC, respectively.
2a: Arrhenius plot Log Vmax Vs 1/ Temperature (K-1) at pH8.0
2b: Florescence analysis to study the thermal behaviour of TALipC
Florescence studies were performed by fluorospectrophotometer (Varian, Inc. Hansen Way, Palo
Alto, CA, USA) where 292 nm was used for excitation and 300 to 550 nm for emission.
3: Kinetics parameter of TALipC studied on the hydrolysis of p-NP palmitate
4: GC chromatogram showing fatty acid selectivity during hydrolysis of an equimolar
mixture of triacylglyceride mixture for a period of 10 min (a) and 30 min (b)
Equimolar triacylglyceride mix (10 mM) [tricaprylin (8:0); tricaprin (10:0); trilaurin (12:0);
trimyristin (14;0); tripalmitin (16:0)] was made in 2-propanol + 50mM pH 8.0 buffer and
reaction was started with the addition of enzyme to make the final volume of 2.0 ml, where final
concentration of substrate become 5 mM. The reaction mixture was incubated at 50 °C for
various time intervals (10 min, 30 min). Reaction was terminated by adding 500 µl of 3M
calcium chloride and centrifuged at 10,000 g for 5 min. The pellet was recovered and dissolved
in hexane to be analysed by gas chromatography by using capillary column using conditions
were: Temp – 250 ⁰C, pressure – 126.6 Kpa, total flow – 150.0 ml/min, column flow – 2.87
ml/min, linear flow – 50.9 cm/sec. Peak a and b corresponds to lauric acid and myristic acid
having retention time 17.5 min and 20.8 min respectively, as per standards.
5: Solvent stability of TALipC
100% activity = 914 U/mg
6: Effect of various metal ions on the activity of lipase
The enzyme was incubated in metal ions 20 mM for 1 h and assayed for lipase activity. Residual
activity was calculated with respect to untreated enzyme as control (100 % = 915 U/mg)
Supplementary Fig. 1a
Supplementary Fig. 1b
Supplementary Fig. 1c
Supplementary Fig. 2a
Supplementary Fig. 2b
400
Intensity at 380 nm
350
300
250
200
150
0
20
40
60
Temperature (°C)
80
100
Supplementary Fig. 3
11
10
3
1/V X10 umole/mg/min
9
8
7
6
5
4
3
2
1
0
-30 -25 -20 -15 -10 -5 0
5
10 15 20 25 30 35 40 45 50 55
1/[S] X 10 -3(mM)
Supplementary Fig. 4a
a
Supplementary Fig. 4b
a
b
Supplementary Fig. 5
180
160
Relative activity (%)
140
120
100
80
60
40
20
0
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Solvents
Supplementary Fig. 6
180
160
Relative activity (%)
140
120
100
80
60
40
20
0
Ba2+
Ca2+
Mg2+
Mn2+
Zn2+
20 mM metal ions
Cd2+
Hg2+