เอกสารการประชุมวิชาการและเสนอผลงานวิจัย มหาวิทยาลัยทักษิณ ครั้งที่ 19 2552 - page 928

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„µ¦ž¦³»¤ª·µ„µ¦Â¨³Áœ°Ÿ¨Šµœª·‹´¥¤®µª·š¥µ¨´¥š´„¬·– ‡¦´Ê Šš¸É 19 ž¦³‹Îµže 2552
‹µ„œ´Ê œšÎµ„µ¦Â¥„Á°œÅŽ¤rÁŽ¨¨¼Á¨Â¨³Ãž¦˜· Á°ÄœœÊ室´„Áž¨º°„´ž³¦—ץčo°´˜¦µnªœ °Š PEG
1500 15% ¨³Á„¨º° Ammonium sulphate 19.2% ¨o ªœÎµ´Êœ¡°¨·Á¤°¦r¨³´ÊœÁ„¨º°¤µšÎµ„µ¦„ε‹´—¡°¨·Á¤°¦r¨³
Á„¨º°°°„—oª¥Áš‡œ·‡ Gel filtration ץčo Sepharose 6B œµ—‡°¨´¤œr 30 cm × 2 cm ¦³´¢Á¢°¦rš¸Éčo‡º°
50 mM Tris-HCl pH 7.0 ¨³Äo°´˜¦µ„µ¦Å®¨Ášnµ„´ 0.22 ml/min Á„ȝ Fraction ¨³ 2 ml Ž¹ÉŠÅ—o—ŠÅªo—´Š£µ¡š¸É 2
¨³ 3 ‹µ„Ÿ¨„µ¦«¹ „¬µÂ—ŠÄ®oÁ®Èœªnµ Á¤ºÉ°œÎµµ¦¨³¨µ¥´Êœ PEG 1500 ޚε Gelfiltration ‹³µ¤µ¦™Â¥„Á°œÅŽ¤r
Þ¦˜·Á°°°„‹µ„ PEG ŗo ×¥ PEG ‹³°°„¤µ„n°œÁœºÉ°Š‹µ„¤¸œµ—Ä®n„ªn µ (Fraction š¸É 34-49) nªœÁ°œÅŽ¤r
Þ¦˜·Á°‹³°°„¤µš¸®¨´Š ÁœºÉ °Š‹µ„¤¸œµ—Á¨È„„ªnµ (Fraction š¸É 50-52) ˜nÁ¤ºÉ°šÎµ„µ¦®µž¦· ¤µ–Á°œÅŽ¤rÁŽ¨¨¼Á¨Äœ
´Êœ PEG ¡ªnµ Ťn¡ž¦·¤µ–Á°œÅŽ¤rÁŽ¨¨¼Á¨š´ÊŠš¸É˜¦ª‹¡ž¦·¤µ–Á°œÅŽ¤rÁŽ¨¨¼Á¨µŠn ªœÄœ´Êœ PEG „n°œšÎµ
Gel filtration °µ‹Áž}œÁ¡¦µ³„µ¦šÎ µ Gel filtration Áž}œ„µ¦šÎµÄ®oÁ°œÅŽ¤rÁŽ¨¨¼Á¨š¸É¤¸Á¨È„œo°¥™¼„Á‹º°‹µŠ ‹¹ŠšÎµÄ®o
˜¦ª‹Å¤n¡ž¦· ¤µ–Á°œÅŽ¤r ÁŽ¨¨¼Á¨Äœ´Ê œ PEG ¨³Á¤ºÉ°œÎµµ¦¨³¨µ¥´Ê œÁ„¨º° Ammonium sulphate ޚε
Gelfiltration ‹³µ¤µ¦™Â¥„Á°œÅŽ¤r ÁŽ¨¨¼Á¨°°„¤µÅ—oĜ Fraction š¸É 30-37 ¨³Á¤ºÉ°šÎµ„µ¦®µž¦· ¤µ–Á°œÅŽ¤r
Þ¦˜·Á°Äœ´Êœµ¦¨³¨µ¥Á„¨º° Ammonium sulphate ¡ªnµ Ťn¡ž¦·¤µ–Á°œÅŽ¤rÞ¦˜·Á°š´ÊŠš¸É˜¦ª‹¡ž¦·¤µ–
Á°œÅŽ¤r Þ¦˜·Á°µŠn ªœÄœ´Êœµ¦¨³¨µ¥Á„¨º° Ammonium sulphate „n°œšÎµ Gel filtration °µ‹Áž}œÁ¡¦µ³„µ¦šÎµ
Gel filtration Áž}œ„µ¦šÎµÄ®oÁ°œÅŽ¤r Þ¦˜·Á°š¸É¤¸°¥¼nÁ¨È„œo°¥™¼„Á‹º°‹µŠÁnœÁ—¸ ¥ª„´Á°œÅŽ¤rÁŽ¨¨¼Á¨Äœµ¦¨³¨µ¥
´Êœ PEG —´Šœ´Êœ Áš‡œ·‡ Aqueous two-phase system š¸Éčo Polyethylene glycol 1500 (PEG 1500) 15% ¨³ÄoÁ„¨º°
Ammonium sulphate 19.2% µ¤µ¦™Â¥„Á°œÅŽ¤rÁŽ¨¨¼Á¨°°„‹µ„Á°œÅŽ¤rÞ¦˜·Á°ÄœœÊ室´„Áž¨º°„ž³¦—Å—o
¨³Á¤ºÉ °µ¦¨³¨µ¥´Êœ PEG ¨³´ÊœÁ„¨º°¤µšÎµ Gel filtration ץčo Sepharose 6B ‹³µ¤µ¦™„ε‹´— PEG ¨³Á„¨º°
°°„‹µ„Á°œÅŽ¤rŗo ץŗoŸ¨—´Š˜µ¦µŠš¸É 2 Ž¹É ŠÂ—ŠÄ®oÁ®ÈœÁ°œÅŽ¤r Þ¦˜·Á°š¸É¡Äœ´Êœ PEG Á¤ºÉ°Ÿnµœ‡°¨´¤œr
Sepharose 6B ¨³‹³¤¸‡ªµ¤¦· »š›·ÍÁ¡·É¤ ¹ÊœÁž}œ 3.83 Ášnµ ¨³¤¸ %Yield Ášnµ„´ 48.45% nªœÁ°œÅŽ¤rÁŽ¨¨¼Á¨š¸É
¡Äœ´Ê œÁ„¨º° Á¤ºÉ°Ÿnµœ‡°¨´ ¤œr Sepharose 6B ¨³‹³¤¸‡ªµ¤¦· »š›·ÍÁ¡·É¤ ¹ÊœÁž}œ 2.71 Ášnµ ¨³¤¸ %Yield Ášnµ„´
64.04% ‹¹ Šµ¤µ¦™¦»žÅ—oªn µ „µ¦Â¥„Á°œÅŽ¤rÁŽ¨¨¼Á¨Â¨³Ãž¦˜· Á°ÄœœÊ室´ „Áž¨º°„´ž³¦——o ª¥Áš‡œ·‡
Aqueous two-phase system ¨³šÎµÁ°œÅŽ¤rÄ®o¦· »š›·Í—oª¥„µ¦šÎµ Gel filtration ץčo Sepharose 6B ‹³µ¤µ¦™
Â¥„Á°œÅŽ¤rš´Ê Š°Šœ·—°°„‹µ„„´œÅ—o ¨³šÎ µÄ®oÁ°œÅŽ¤r š¸Éŗo¤¸‡ªµ¤¦· »š›·Í Á¡·É¤¤µ„ ¹Êœ Á¤ºÉ°Áš¸¥„´œÊÎ µ®¤´„
Áž¨º°„´ ž³¦—š¸É¤¸Á°œÅŽ¤rš´ÊŠ°Šœ· —œ¸Ê°¥¼n¦nª¤„´œ
˜µ¦µŠš¸É 2 —ŠŸ¨„µ¦šÎµ¦· »š›·ÍÁ°œÅŽ¤rÁŽ¨¨¼Á¨Â¨³Ãž¦˜· Á°ÄœœÊ室´„Áž¨º°„´ž³¦—
Sample Protein
(mg/ml)
Protease Cellulase
Activity
(Units/ml)
Specific
activity
(Unit’s/mg)
%Yield Purification Activity
(Units)
Specific
activity
(Unit’s/mg)
%Yield Purification
œÊ室´„ 13.39 11.25 0.84 100 1 124.24 9.28 100 1
´Êœ PEG 5.31 9.67 1.82 86.01 2.17 3.76 6.64 42.14 0.41
´ÊœÁ„¨º° 7.67 1.89 0.25 16.79 0.29 92.56 12.07 74.50 1.30
Gel filtration
- ´Êœ PEG
- ´ÊœÁ„¨º°
1.69
3.17
5.45
-
3.22
-
48.45
-
3.83
-
-
79.56
-
25.11
-
64.04
-
2.71