Ln

Gly Cys

Figure 2.11 Schematic representation of modified proglycinin A1aB1b. (a) variable region-deleted mutants; (b) disulfide bond-deleted mutants. Numbers and S-S indicate the residue number from N-terminus and a disulfide bond, respectively. (From: Kim, C.S., S. Kamiya, T. Sato, S. Utsumi, M. Kito, Protein Eng. 3:725-731, 1990.)

Gly Cys

Figure 2.11 Schematic representation of modified proglycinin A1aB1b. (a) variable region-deleted mutants; (b) disulfide bond-deleted mutants. Numbers and S-S indicate the residue number from N-terminus and a disulfide bond, respectively. (From: Kim, C.S., S. Kamiya, T. Sato, S. Utsumi, M. Kito, Protein Eng. 3:725-731, 1990.)

gel forming ability and gel hardness similar to those of the native glycinin. On the other hand, AI, IV+4Met, V+4Met, C88S, F163C, N116C, P248C, and N116C/P248C formed harder gels than the normal one did. C88S and N116C/P248C showed the most prominent results. On the other hand, R161C formed harder gels than normal at protein concentrations higher than 7%, but formed gels with gel hardness similar to that of normal to one at protein concentration lower than 7%. C12G formed gels with gel hardness similar to that of normal glycinin at higher protein concentration, but could not form gel at a protein concentration lower than 5.6%. These results indicate: (1) the disulfide bond between Cys12 and Cys45 plays an important role in the initiation of a disulfide exchange reaction for a gelation; (2) introduction of a new sulfhydryl group and a disulfide bond is useful for improvement of heat induced gel forming ability and gel hardness; (3) the reactivity of the disulfide bond between Cys12 and Cys45 of AI, where its N-terminus is Cys12, increases; and (4) enhancement of surface hydrophobicity may improve gel forming ability.

We compared the emulsifying ability of AI, AV8, IV+4Met, V+4Met, C12G, C88S, and D157A with that of proglycinin A1aB1b. AI, IV+4Met, C12G, and C88S exhibited similar emulsifying ability to that of proglycinin A1aB1b. D157A exhibited worse emulsifying ability than proglycinin A1aB1b did (the details will be described elsewhere). In contrast, AV8 and V+4Met exhibited better emulsifying ability. These results indicate: (1) the presence of a hydrophobic region at the C-terminus increases emulsifying ability; (2) the effect of increasing hydrophobicity is different among the mutants; and (3) a destabi-lization of a structure does not have an effect on the improvement of the emulsifying ability of proglycinin A1aB1b. These observations correspond to the results of the analyses of glycinin having different subunit compositions described in the previous section.

Protein engineering is useful not only for the improvement of physicochemical functions, but also to elucidate the relationship between structure and physicochemical function. Therefore, the elucidation of the relationship between structure and physico-chemical function at a molecular level, by protein engineering, will enable us to improve the physicochemical functions of soybean proteins effectively and rationally.

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