Synthesis of thermo-sensitive copolymer with affinity butyl ligand and its application in lipase purification.
In this study, thermo-sensitive N-alkyl substituted polyacrylamide polymer P(NNB) was synthesized by using N-hydroxymethyl acrylamide(NHAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers, and its low critical solution temperature (LCST) was controlled to be 28 degrees C. The recovery of the thermo-sensitive polymer was over 98%. Butanol as a hydrophobic ligand was covalently attached onto polymer P(NNB) and butyl ligand density was 80 micromol g(-1) polymer. The affinity polymer was used for purification of lipase from crude material. Optimized condition was pH 7.0, 35 degrees C adsorption temperature, 120 min adsorption time and 0.5 mg ml(-1) initial concentration of lipase. The adsorption isotherm accords with a typical Langmuir isotherm. The maximum adsorption capacity (Q(m)) of the affinity polymer for lipase was 24.8 mg g(-1)polymer. The affinity copolymer could be recycled by temperature-inducing precipitation and there was only about 6% loss of adsorption capacity after five recyclings. Specific activity of lipase was improved from 14IU mg(-1) to 506 IU mg(-1) protein, and its recovery achieved 82%. The affinity polymer is suitable for the purification of target proteins from the crude material with large volume and dilute solution.
Zhou
Libo
L
State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science & Technology, 130 Meilong Rd., Shanghai 200237, China.
Wan
Junfen
J
Cao
Xuejun
X
D000178:Acrylamides / Q000737:chemistry; D000179:Acrylates / Q000737:chemistry; D000180:Acrylic Resins / Q000737:chemistry*; D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D013696:Temperature; D000099166:Polyacrylamides
D000178:Acrylamides; D000179:Acrylates; D000180:Acrylic Resins; D008049:Lipase; D000099166:Polyacrylamides; C032490:n-butyl acrylate; C067295:N-isopropylacrylamide; C066443:N-methylolacrylamide