화학공학소재연구정보센터
Enzyme and Microbial Technology, Vol.26, No.8, 582-592, 2000
A systematic approach to the large-scale production of protein crystals
Crystallization has recently emerged as a suitable process for the manufacture of biocatalysts in the form of cross-linked enzyme crystals (CLECs(TM)) or for the recovery of proteins from fermentation broths. In both instances it is essential to define conditions which control crystal size and habit, and that yield a reliable recovery of the active protein. Experiments to define the crystallization conditions usually depend on a factorial design (either incomplete or sparse matrix) or reverse screening techniques. In this work, we describe a simple procedure that allows the effect of three factors, for example protein concentration, precipitant concentration and pH, to be varied simultaneously and smoothly over a wide range. The results are mapped onto a simple triangular diagram where a 'window of crystallization' is immediately apparent, and that conveniently describes variations either in the crystal features, such as their yield, size, and habit, or in the recovery of biological activity. The approach is illustrated with two enzymes, yeast alcohol dehydrogenase (ADH Ij and Candida rugosa lipase. For ADH the formation of two crystal habits (rod and hexagonal) could be controlled as a function of pH (6.5-10) and temperature (4-25 degrees C). At pH 7, in 10 to 16% w/v polyethylene glycol (PEG) 4000, only rod-shaped crystals formed whereas at pH 8, in 10 to 14% w/v PEG, only hexagonal crystals existed. For both enzymes, catalyst recovery was greatest at high crystallization agent concentrations and low protein concentration. For ADH, the greatest activity recovery was 87% whereas for the lipase crystals, by using 45% v/v 2-methyl-2,4-pentanediol (MPD) as the crystallization agent, a crystal recovery of 250 crystals per mu l was obtained. For the lipase system, the use of crystal seeding was also shown to increase the crystal recovery by up to a factor of four. From the crystallization windows, the original conditions based on literature precedent (35% v/v MPD, 1 mM CaCl2, 1.8 mg protein/ml) were altered (47.5% v/v MPD, 2 mM CaCl2, 3 mg protein/ml). This led to an improved recovery of the lipase under conditions that scale reliably from 0.5 mi to 500 mi with no change in size, shape or recovery of the crystals themselves. Finally, these crystals were crosslinked with 5% v/v glutaraldehyde and mass and activity balances were calculated for the entire process of CLEC(TM) production. Up to 35% of the lipase activity present in the crude solid was finally recovered in the lipase CLECs(TM) after propan-2-ol fractionation, crystallization, and crosslinking. (C) 2000 Elsevier Science Inc. All rights reserved.