Lee, Timothy Shan Wei; (2000) The effect of crystal form on the catalytic and mechanical stability of cross-linked enzyme crystals (CLECs). Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cross-linked enzyme crystals (CLECs) are a novel form of immobilised biocatalyst which exhibit higher catalytic stability than conventional immobilised enzyme preparations. This makes them suitable for application in industrial biotransformation processes and for the synthesis of chiral pharmaceuticals. In this work, a new and generic screening technique for protein crystallisation is first described which allows the effect of three factors, e.g. protein concentration, precipitant concentration and pH, to be varied simultaneously. The results are presented as a simple two-dimensional triangular diagram where a 'window of crystallisation' describing activity recovery, crystal size etc. can be immediately visualised. The approach is illustrated with yeast alcohol dehydrogenase I (YADH I). The formation of two crystal habits (rod and hexagonal) could be controlled as a function of pH (6.5-10) and temperature (4-25 °C). At pH 7, in 10-16% (w/v) polyethylene glycol (PEG) 4000, only rod-shaped crystals (4.6 μm) formed while at pH 8, in 10-14% (w/v) PEG, only hexagonal crystals (10 μm) existed. Catalyst recovery was greatest (87%) at high crystallisation agent concentrations and low protein concentration. YADH I crystallisation was successfully scaled up from 0.5 to 500 mL (1,000 fold) with reproducible results, in terms of crystal size, shape and recovery being obtained at both scales. Both crystal forms of YADH I were cross-linked with glutaraldehyde to form CLECs. Catalytic studies showed that hexagonal CLECs maintained a more constant specific activity at higher temperatures (50 °C) and within a broader pH range of 5-11 than rod CLECs. Hexagonal CLECs also maintained a constant activity up to 24 hours in the presence of proteases as opposed to rod CLECs which lost activity after 6 hours. Rod CLECs however were catalytically more stable than hexagonal CLECs in aqueous-organic solvents (log P values from -0.76 to 3), maintaining greater than 80% of their initial activity. Small-scale experiments using a rotating-disc shear device were performed as a function of speed and duration of disc rotation and over a range of CLEC concentrations (0.26-2.5 mg.mL-1) and energy dissipation rates (2.2*10^3-6.8*10^5 W.kg-1). Owing to their small size, no breakage occurred for rod CLECs. Breakage of the larger hexagonal-shaped CLECs did occur at energy dissipation rates, emax, above 1.0*10^5 W.kg-1 where the length scale of turbulence was less than 2.0 μm. Based on visual observation of the sheared CLEC suspensions and models of crystal breakage, it was concluded that breakage of the hexagonal CLECs occurred due to shear-induced attrition. Small-scale filtration studies showed that rod-shaped CLECs had a greater flux and hence lower cake resistance (3-9*10^12 m.kg-1) than the flat hexagonal CLECs (1-2*10^14 m.kg-1). The specific cake resistances of rod and hexagonal CLECs remained constant during several filtration cycles at 50-100 kPa. Size measurements indicated that no breakage of crystal forms occurred as a result of pressure. The rotating-disc shear device was shown to be a useful scale-down tool to predict the breakage of CLECs by comparing the maximum shear rate, Gmax, of 3.4*10^5 s-1 to that found in a standard Rushton turbine reactor. It was predicted that no breakage of CLECs would occur at impeller speeds (200 rpm) sufficient to disperse the crystals. Breakage of CLECs occurred at Gmax values above 1.5x104s-1 corresponding to rotational speed above 1,000 rpm. Breakage of CLECs reduced the recovery of the catalyst due to possible membrane clogging by fines generated by shear-induced attrition.

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