WT Val QGQYKNQDPI ILVHGFNGFT DDINPAVLAH YWGGDKLNIR QDLESNGYET
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....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 WT_Val QGQYKNQDPI ILVHGFNGFT DDINPAVLAH YWGGDKLNIR QDLESNGYET Mut_Lys QGQYKNQDPI ILVHGFNGFT DDINPAVLAH YWGGDKLNIR QDLESNGYET Mut_Asp QGQYKNQDPI ILVHGFNGFT DDINPAVLAH YWGGDKLNIR QDLESNGYET Clustal Co ********** ********** ********** ********** **********
....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 WT_Val YEASVGALSS NYDRAVELYY YIKGGTVDYG AAHAEKYGHE RYGKTYEGVY Mut_Lys YEASVGALSS NYDRAVELYY YIKGGTVDYG AAHAEKYGHE RYGKTYEGVY Mut_Asp YEASVGALSS NYDRAVELYY YIKGGTVDYG AAHAEKYGHE RYGKTYEGVY Clustal Co ********** ********** ********** ********** **********
....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 WT_Val KDWQPGKKVH LVAHSMGGQT VRQLEELLRN GNQEEIEYQK EHGGEISPLF Mut_Lys KDWQPGKKVH LVAHSMGGQT VRQLEELLRN GNQEEIEYQK EHGGEISPLF Mut_Asp KDWQPGKKVH LVAHSMGGQT VRQLEELLRN GNQEEIEYQK EHGGEISPLF Clustal Co ********** ********** ********** ********** **********
....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 WT_Val QGNNDNMVNS ITTIGTPHNG THAADALGNE AIVRQLAFDY AKFKGNKNSK Mut_Lys QGNNDNMVNS ITTIGTPHNG THAADALGNE AIVRQLAFDY AKFKGNKNSK Mut_Asp QGNNDNMVNS ITTIGTPHNG THAADALGNE AIVRQLAFDY AKFKGNKNSK Clustal Co ********** ********** ********** ********** **********
....|....| ....|....| ....|....| ....|....| ....|....| 210 220 230 240 250 WT_Val VDFGFGQWGL KQREGETYAQ YVQRVQNSGL WKTEDNGFYD LTREGAAKLN Mut_Lys VDFGFGQWGL KQREGETYAQ YVQRVQNSGL WKTEDNGFYD LTREGAAKLN Mut_Asp VDFGFGQWGL KQREGETYAQ YVQRVQNSGL WKTEDNGFYD LTREGAAKLN Clustal Co ********** ********** ********** ********** **********
....|....| ....|....| ....|....| ....|....| ....|....| 260 270 280 290 300 WT_Val KNTSLNPNIV YKTYTGESTR PTLFGNQKSD VNLFLPFTVT GNVIGKAAEK Mut_Lys KNTSLNPNIV YKTYTGESTR PTLFGNQKSD VNLFLPFTVT GNVIGKAAEK Mut_Asp KNTSLNPNIV YKTYTGESTR PTLFGNQKSD VNLFLPFTVT GNVIGKAAEK Clustal Co ********** ********** ********** ********** **********
....|....| ....|....| ....|....| ....|....| ....|....| 310 320 330 340 350 WT_Val EWRENDGLVS TISSQHPFNQ AFIEATDEVK KGVWQVTPIK HGWDHVDFVG Mut_Lys EWRENDGLKS TISSQHPFNQ AFIEATDEVK KGVWQVTPIK HGWDHVDFVG Mut_Asp EWRENDGLDS TISSQHPFNQ AFIEATDEVK KGVWQVTPIK HGWDHVDFVG Clustal Co ******** * ********** ********** ********** **********
....|....| . 360 WT_Val QDSTDSNHPT E Mut_Lys QDSTDSNHPT E Mut_Asp QDSTDSNHPT E Clustal Co ********** *
Figure 1. Aminoacid alignement of S. xylosus recombinant lipase (WT-Val) and mutants (Mut-Asp and Mut-Lys). Catalytic triade was shown is red, mutated aminoacids were underlined. 1 2 3 4 5 6 7 8 75 50 37 25
Figure 2. SDS-PAGE of recombinant S. xylosus lipases purification by nickel resin. Lane 1 Molecular weight markers, lane 2, BSA; lanes 3 and 4, WT-Lip; lanes 5 and 6, Mut-Asp; lanes 7 and 8 Mut-Lys.