Supplementary Tables

Table 1. Primers used to amplify lipase genes LIP8, LIP14 and LIP18 from Y.lipolytica MSR80

Primers / Sequences (5’ – 3’) / Restriction enzyme
lip8-F / GAATTCTGTGTCTCAAGGG / EcoRI
lip8-R / AAGCTTTCAGTTCTCAACTTG / HindIII
lip14-F / GAATTCTTCTCCCCTTACAG / EcoRI
lip14-R / AAGCTTCTAAAAGATGGTGGA / HindIII
lip18-F / GAATTCGGCGTTAAGTAGT / EcoRI
lip18-R / AAGCCTTTAGTCTATTTGCAC / HindIII

Table 2.In silico analysis of Yarrowialipolytica

SNo. / Gene I.D / Common name / Protein length(a.a) / Molecular weight (Da) / Isoelectric point / Secretory pathway
1 / YALIOA20350g / YILIP-2 / 334 / 36784 / 6.1 / Extracellular
2. / YALIOBO8030g / YILIP-3 / 498 / 55872.7 / 6.2 / Unknown
3. / YALIOBO9361g / YILIP-8 / 371 / 41422.8 / 6.1 / Extracellular
4. / YALIOB11858g / Unknown / 349 / 38816.9 / 5.4 / Extracellular
5. / YALIOB20350g / Unknown / 376 / 42663.7 / 7.5 / Extracellular
6 / YALIOC00231g / Similar to YILIP-1 / 478 / 53657.1 / 5.3 / Unknown
7. / YALIOC19580g / Unknown / 380 / 42471.9 / 9.8 / Unknown
8. / YALIODI9064g / Unknown / 424 / 47076.7 / 6.6 / Extracellular
9. / YALIOD15906g / Unknown / 324 / 36210.2 / 6.4 / Extracellular
10. / YALIOD17534g / Unknown / 666 / 75828.2 / 5.9 / Extracellular
11. / YALIOD19184g / YILIP-7 / 366 / 40849.2 / 6.0 / Extracellular
12. / YALIOE00286g / Unknown / 317 / 36219.3 / 6.1 / Extracellular
13. / YALIOE00528g / Unknown / 504 / 58276.6 / 7.9 / Unknown
14. / YALIOE02640g / YILIP-5 / 370 / 40255.4 / 6.4 / Extracellular
15. / YALIOE03806g / Unknown / 244 / 27480 / 5.4 / Unknown
16. / YALIOE05995g / YILIP-1 precursor / 476 / 53084.7 / 5.6 / Unknown
17. / YALIOEO8492g / YILIP-4 / 406 / 46339.4 / 6.3 / Unknown
18. / YALIOE10659g / YILIP-1 / 486 / 55381.6 / 4.8 / Unknown
19. / YALIOE11561g / Unknown / 406 / 46057.4 / 6.6 / Extracellular
20. / YALIOE31515g / Unknown / 327 / 37272.4 / 7.4 / Unknown
21. / YALIOE32035g / Unknown / 569 / 63587.4 / 7.6 / Extracellular
22. / YALIOE34507g / Unknown / 412 / 45684.4 / 6.6 / Extracellular
23. / YALIOF10010g / Unknown / 816 / 91097.2 / 7.4 / Extracellular
24. / YALIOF11429g / Unknown / 290 / 32475.5 / 5.7 / Extracellular
25. / YALIOF19030g / Unknown / 823 / 82657.7 / 4.6 / Extracellular

Table 3a. Effect of inhibitors on recombinant lipases

Inhibitors (10mM) / LIP8
Relative activity % / LIP14
Relative activity % / LIP18
Relative activity %
Control / 100 (117 U/mg) / 100 (119 U/mg) / 100 (111 U/mg)
EDTA / 94.8 ± 3.2 / 84.7 ± 5.1 / 89.1 ± 2.8
ME / 185.7 ± 5.5 / 190.2 ± 7.2 / 196.5 ± 3.7
DTT / 159.3 ± 7.3 / 144.7 ± 1.3 / 172.7 ± 6.2
1,10-Phenanthraline / 52.7 ± 5.2 / 48.3 ± 4.1 / 42.4 ± 2.6
DTNB / 33.4 ± 6.9 / 24.6 ± 3.9 / 12.7 ± 6.0
PMSF / 23.3 ± 1.2 / 16.1 ± 2.0 / 8.4 ± 1.1
N-Bromosuccinamide / 10.3 ± 2.3 / 6.2 ± 4.0 / 1.1 ± 3.2

Table 3b. Effect of solvents on the activity of lipases

Solvents
90% (v/v) / LIP8
Relative activity % / LIP14
Relative activity % / LIP18
Relative activity %
Control / 100 (117 U/mg) / 100 (119 U/mg) / 100% (111 U/mg)
Ethanol / 8.5 ± 1.2 / 7.9 ± 2.4 / 3.2 ± 1.2
Butanol / 2.2 ± 1.1 / 25.2 ± 8.2 / 0.8 ± 1.9
Iso-propanol / 66.9 ± 7.5 / 40.9 ± 5.4 / 98.2 ± 6.2
Acetonitril / 104.2 ± 4.3 / 109.4 ± 6.1 / 71.1 ± 5.3
Ethyl acetate / 89.9 ± 3.8 / 83.0 ± 9.1 / 76.6 ± 1.8
Diethyl ether / 200.0 ± 9.2 / 190.0 ± 7.7 / 87.8 ± 6.4
DMSO / 52.8 ± 7.2 / 49.5 ± 8.6 / 33.3 ± 5.5
1,4-dioxane / 237.4 ± 3.2 / 251.2 ± 5.9 / 192.0 ± 4.4
Hexane / 207.0 ± 6.1 / 210.0 ± 4.7 / 73.8 ± 3.2

Table 4. Fatty acid composition of oils

Oils / Composition / Inference
Olive oil / C18:1 65-80%, C18:2 10% / Rich in oleic acid
Ground nut oil / C18:1 52-60%, C18:2 13-27% / Rich in oleic acid
Cotton seed oil / C16 20%, C18:1 35%, C18:2 42% / Rich in linoleic acid
Corn oil / C18:1 19-49%, C18:2 34-62% / Rich in linoleic acid
Amla oil / C18:1 20-40%, C18:2 60-80% / Rich in linoleic acid

Supplementary figure legends

Fig. 1.Multiple sequence alignment of LIP8, LIP14 and LIP18 from Y.lipolyticaMSR80

conserved sequence are marked by box and active residues are marked by circle

Fig. 2.Effect of pH in the activity of LIP14, LIP18 and LIP8

Different buffers with the strength of 50mM were used ranging from pH 3.0 to pH 11.0 in which Citrate-phosphate buffer ranges from pH 3.0-7.0, Potassium phosphate buffer pH 8.0, Tris/HCl buffer pH 9.0 and Sodium carbonate-Bicarbonate pH 10.0, Glycine-NaOHbuffer pH 11.0. pH optima was determined by performing enzyme assay at 40°C in different buffers using p-np-palmitate .The enzyme unit was calculated using their respective extinction coefficients for p-nitrophenol at different pH.

Fig. 3.Thermal behaviour of lipases LIP14, LIP18 and LIP8

Lipases were incubated at different temperatures ( 40 °C, 50 °C, 60 °C, 70 °C, 80°C) for varying time (10 min, 30 min, 60 min, 90 min and 120 min) and activity was calculated using standard protocol at 40 °C, pH 7.0)

Fig. 4. Effect of metal ions on LIP8, LIP14 and LIP18

100 % activity for LIP8, LIP14 and LIP18 corresponds to 117 U/mg, 119 U/mg, 111 U/mg on p-np-palmitate

Supplementary figures

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