Abstract
The extracellular lipase Yarrowia lipolytica (YLLIP2) crude extract was efficiently separated and purified from Candida sp. 99–125 by one-step ion-exchange chromatography on polyethyleneimine (PEI) functionalized monolithic columns. The preparative conditions for the functionalization of monoliths were optimized, including PEI molecular mass, PEI concentration, modification time and temperature. The monolithic skeleton was prepared in situ by polymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) with a volume ratio of 8:2. Heptane was used as the porogen. PEI 30 kDa with the concentration of 10% (v/v) was applied for the modification of the monolith at 55 °C for 12 h. Lipase (EC.3.1.1.3) from Candida sp. 99–125 was separated to four isoforms (isoform A, isoform B, isoform C and isoform D). As analyzed on non-denaturing PAGE and MALDI-TOF–MS, the four isoforms are homogenous and have the same molecular mass of approximate 38 kDa. The monoliths can afford direct crude lipase loading without increasing too much back pressure, which explores the great potential of the application of monoliths for one-single step fast separation and purification of complicated proteins.
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