Purification of proteolytic enzymes and their effects on rheological changes on cracker sponges: A study of wheat hardness using electrophoresis

• Main Author: Lin, Wei-Daw Alfred
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1991
• Subjects: Agricultural engineering; Chemical engineering
• ISBN: 9798207481388

Enzymes were extracted from wheat flours with ammonium sulfate. The proteolytic activity of extracted enzymes was measured with a fluorometric method. The pH optimum of the extracted enzymes on hemoglobin substrate was around pH 4.0. The ratio of endo- to exo-proteolytic enzymes appeared to be similar in extracts from both soft and hard wheats. However, soft wheat flours had higher proteolytic activity than did hard wheat flours. The enzymes were purified by gel filtration chromatography. The presence of two peaks of proteolytic activity was detected. Lubricated uniaxial compression was used to measure the changes of elongational viscosity of the cracker sponges set at pH 4 during fermentation. It was found that the elongational viscosity of the sponges decreased with fermentation time. Insignificant changes of the elongational viscosity of the sponges were observed when enzymes had been extracted from sponge flour. The elongational viscosity of the sponge decreased with fermentation time when the extracted enzymes were added back to the flour residue. One of the active fractions eluted from G-100 Sephadex was responsible for the change of the elongational viscosity of the sponge during fermentation. Rechromatography was used to further purify the proteolytic enzymes, where single peak with high specific proteolytic activity was obtained. Starch was isolated by two different methods, dough-forming and slurry method. By using the dough-forming method, starch granule proteins (SGP) from different genetic backgrounds of starch were extracted with 0.1 M NaCl at 25$\sp\circ$C for 20 min or 1% sodium dodecyl sulfate at 50$\sp\circ$C for 20 min. The protein distribution of SGP was examined by SDS-PAGE. Soft wheats showed constantly a strong 15 KD electrophoretic band; whereas, hard wheats gave a relatively weak electrophoretic band, and durum wheat had no band. The data support the results of Greenwell and Schofield (1986) that the 15 KD electrophoretic band is associated with wheat softness. However, an equivalent amount of a 15 KD electrophoretic protein band was found for both hard and soft wheat when the starch was prepared by the slurry method. Both pronase and sodium dodecyl sulfate effectively removed S6P from the surface of starch granules.