Sorbitol, enzyme stabilization

This effect of polyhydroxyl compounds may not be quite as simple as it has been described, as the structure of the polyhydroxyl compound may play some part in effective stabilization of enzymes in wet systems. Thus Fujita et al, (20) reported that inositol was more effective than sorbitol in stabilizing lysozyme in aqueous solutions. Both compounds contain six hydroxyl groups, but inositol is cyclic in structure whereas sorbitol is linear, Fig 10. The interaction of polyhydroxyl compounds with water promotes a change in the molecular structure of water. Inositol was reported to have a larger structure-making effect than sorbitol, which accounted for the greater stabilization effect of this compound. 

An improvement over this earlier system was attained with the addition of boron compounds such as boric acid or borate salts [106-108], It has been hypothesized that boric acid and calcium form intramolecular bonds which effectively crosslink or staple an enzyme molecule together [107,108], The use of polyols such as propylene glycol, glycerol, and sorbitol in conjunction with the boric acid salts further enhances the stability of these enzymes [109-111], The patent literature contains numerous examples of enzyme stabilization systems that utilize borates, polyols, carboxylate salts, calcium, and ethanolamines, or combinations thereof [91,112-115],... 

Among the three enzymes participating in L-cysteine production, l-ATC hydrolase was found to be the least stable11301. However, the stability of l-ATC hydrolase was sharply enhanced as water activity decreased from 0.93 to 0.80. In the absence of sorbitol, the stability of l-ATC hydrolase increased in proportion to ionic strength. Thus, Ryu et al. succeeded in enhancing the half life of l-ATC hydrolase by 10-fold to 20-fold in sorbitol-salt mixtures11301. 

A second approach to efficient synthesis of gluconic acid and sorbitol has been the use of cell-free GFOR from Z. mobilis. Using a crude extract of Z. mobilis in a continuous ultrafiltration membrane reactor, excellent substrate conversion and enzyme stability were maintained for about 10 days (Silva-Martinez et al. 1998). However, it has been concluded that the strict requirements for enzyme stability would be costly for commercial production and downstream processing, which is a strong argument favoring use of permeable cells (Nidetzky et al. 1997). 

Polyalcohols, such as glycerol, sugar, sorbitol, and propylene glycol may prevent denaturation (28). Also substrates or substrate analogues often stabilize by conferring an increased rigidity to the enzyme stmcture. 

A novel nitrilase was purified from Aspergillus niger K10 cultivated on 2-cyanopyridine. It was found to be homologous to a putative nitrilase from Aspergillus fumigatus Af293. The nitrilase exhibited maximum activity at 45 °C and pH 8.0 with much less activity observed at slightly acid pH. Its substrate preference was for 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thio-phen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. ( )-2-Phenylpropionitrile was only poorly converted by this enzyme and with minimal enantioselectivity. The enzyme was shown to be multimeric (>650 kDa) and be stabilized in the presence of sorbitol and xylitol [57]. 

Proteases have received less attention than lipases, but in one of the earliest papers on biocatalysis in ionic liquids it was noted that the activity loss of thermo-lysin during preincubation proceeded much more slowly in [BMIm][PF6] than in ethyl acetate [8]. The storage stability of a-chymotrypsin in the ionic liquid [EMIm][ Tf2N] was compared with that in water, 3 M sorbitol, and 1-propanol. The residual hydrolytic activity (after dilution with aqueous buffer) was measured vs time, and structural changes were monitored by fluorescence and CD spectroscopy as well as DSC [98]. The enzyme s life-time in [EMIm][ Tf2N] at 30°C was more than twice that in 3 M sorbitol, six times as long as that in water, and 96 times as long as that in 1-propanol. 

Furthermore, the stability of a-chymotrypsin in [EMIM][(CF3S02)2N] was studied by De Diego et al. Results were compared to those obtained in 1-propanol, a deactivating medium, and an aqueous solution of sorbitol, an enzyme-stabihzing medium. Using fluorescence and circular dichroism studies they showed that of the solvents used only the ionic liquid was able to stabilize the enzyme via the formation of a flexible and more compact 3D structure [41]. 

Protein stability may be regarded as the opposite of denaturation. The stability of enzymes (and proteins) can be increased in many ways, e.g., by microenvironmental changes, immobilization, and protein engineering (78). Enzymes are more stable in the presence of polyols (ethylene glycol, glycerol, erythritol, and sorbitol), polymers (PEG, dextrans), and carbohydrates (sucrose, lactose, and trehalose). Hydrophilic enzymes are stabilized by the presence of salts (LiCl, NaCl, and KCl), whereas hydrophobic enzymes are hardly affected by salts. Proteins are also stabilized by compounds that bind specifically to the folded conformation. Most of the metalloenzymes and the enzymes that have an anion-binding site fall into this category. 

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