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˜µ¦µŠš¸É 4 —Šž¦·¤µ–Á°œÅŽ¤rŨÁž‡ªµ¤¦· »š›·Í°ŠÁ°œÅŽ¤r Partition coefficient (Klipase) ¨³%Yield ץčo
PEG 6000 ¨³Á„¨º° trisodium citrate
PEG
Mol.Wt.
PEG
(w/v%)
TriNa-citrate
(w/v%)
Top phase
Activity(U/ml)
Bottom phase
Activity(U/ml)
Total
volume(ml)
Klipase Top phase(PEG phase)
SP PF %Yield
6000
5 26 - 0.012 10.0 - - - -
10 20 0.034 0.015 11.0 2.23 0.047 150.55 28.05
15 16 0.024 0.016 11.5 1.46 0.041 131.61 38.89
20 14 0.018 0.019 10.0 0.95 0.034 111.02 36.69
25 12 0.015 0.020 10.5 0.75 0.029 93.20 36.35
30 11 0.013 0.022 11.5 0.56 0.027 85.51 38.90
35 10 0.011 0.024 11.0 0.46 0.022 69.83 33.23
40 9 0.009 0.027 11.5 0.35 0.019 60.54 33.21
S=Salt (bottom) phase, P= PEG (top) phase, PF= Purification Factor, SP= Specific activity(Units/mg protein)
£µ¡š¸É 3 „µ¦Â¥„µ¦„´—®¥µš¸ÉÁ®¨º °‹µ„„µ¦„´—œÊε¤´œ¤³¡¦oµª¦³®ªnµŠ´Êœ PEG (—oµœœ) ¨³´ÊœÁ„¨º°
(—oµœ¨nµŠ) ; PEG 1500 (a) ¨³ PEG 6000 (b)
Ÿ¨„µ¦«¹„¬µ¦¼ žÂÃž¦˜¸œ—oª¥Áš‡œ·‡ SDS-PAGE
„µ¦«¹ „¬µÂ™Ãž¦˜¸œÄœ˜´ ª°¥nµŠš¸ÉŸnµœ„µ¦Â¥„Á°œÅŽ¤rŨÁž—oª¥Áš‡œ·‡ ATPS ‹µ„nªœš¸ÉÁ®¨º °š·ÊŠ
®¨´ Š‹µ„„µ¦„´—Â¥„œÊÎ µ¤´œ¤³¡¦o µª Á¡ºÉ°Áž¦¸¥Áš¸ ¥¨´„¬–³ °ŠÂ™Ãž¦˜¸œš´ÊŠ„n °œÂ¨³®¨´Š‹µ„„µ¦ÄoÁš‡œ· ‡
ATPS ‹³Å—oŸ¨„µ¦š—¨°Š ¨³‡Î µœª–®µœÊ宜´„Þ¦˜¸ œÃ—¥Áž¦¸¥Áš¸¥„´Ãž¦˜¸œ¤µ˜¦“µœ —´Š£µ¡š¸É 4 ‹³¡ªnµ
¦¼žÂÂ™Ãž¦˜¸œ °Š 10 % PEG 1500 ‹³ž¦µ„’™Ãž¦˜¸ œ 3 ™ Ž¹ÉŠµ¤µ¦™‡Îµœª–®µœÊ宜´„äÁ¨„»¨Å—o
‹³Ãž¦˜¸œš¸É¤¸œÊ宜´„Áž} œ 50.12 24.55 ¨³15.14 kDa ˜µ¤¨Îµ—´ ÁœºÉ°Š‹µ„Þ¦˜¸ œ¦ª¤š´ÊŠ®¤—Äœ´Êœ PEG ¤¸œo °¥
„ªn µÃž¦˜¸œÄœ´ÊœÁ„¨º° ¨³µ¦„´—®¥µ¤µ„ ץÞ¦˜¸ œ¦ª¤Äœ´Êœ PEG (10% PEG) ž¦³¤µ– 1.04 mg ˜nĜ
µ¦„´—®¥µ¤¸¤µ„™¹Š 775.32 mg šÎµÄ®o‹ÎµÁž}œ˜o°Š¤¸„µ¦Ã®¨—˜´ª°¥n µŠ °Š´Êœ PEG ¤µ„„ªnµ ‹µ„ o°¤¼¨‹³šÎµÄ®o
š¦µªn µ„µ¦Â¥„Þ¦˜¸œ—oª¥Áš‡œ·‡ ATPS ץčo 10 % PEG 1500 „´Á„¨º° 30 % trisodium citrate ‹³Å—ož¦· ¤µ–
Á°œÅŽ¤rŨÁžš¸ÉŞ°¥¼nĜ´Êœ PEG (—o µœœ) ¤µ„š¸É »— ¨³¤¸ž¦·¤µ–Þ¦˜¸œš¸Éœo °¥„ªnµÄœµ¦„´—®¥µ‹¹ ŠnŠŸ¨