Typescript (photocopy). / Digitized by Kansas Correctional Industries

Guanidine-stable chymoelastase (GSC-Elastase), an enzyme isolated from a commercial protease preparation (Pronase), has been shown to be stable and active in the presence of 6.0 M guanidinium chloride (Si egel , S . , et. al . , J . Biol. Chem. 247:4155, J . Pi of . Chem. 248:3233.) The amino acid sequence around the active site serine residue for the protease is similar to that found for bovine chymotrypsin (Awad, W. M. et.al., J. Biol. Chem. 247:4144). This investigation involved the qualitative comparison of the protein cleavage specificity of GSC-Elastase in the presence and absence of denaturant. This specificity was also compared to that found for a-chymotrypsin for the existence of possible similarities in their actionson protein substrates. S-Carboxymethylated lysozyme (CM-HEL) was hydrolyzed in separate trials which were catalyzed by GSC-Elastase in the presence and absence of 6.0 M guanidinium chloride and by a-chymotrypsin. Two-dimensional peptide maps were made from each of the hydrolysates by performing thin-layer chromatography in one direction followed by electrophoresis in a perpendicular direction. The results of the mapping procedure indicated that the cleavage of protein substrates by GSC-Elastase is reproducible and therefore specific under denaturing conditions as well as in the absence of denaturant. These results also suggested that the protein cleavage specificity of GSC-Elastase was found to be significantly different from that of bovine chymotrypsin.

by Poon Hoi To. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1981. / Bibliography: leaves 40-41.

Myofibrillar proteins, the main components that impart functional properties to muscle foods, can undergo denaturation and aggregation during frozen storage. The overall objective of this research was to study the changes in protein structure that are associated with the preparation and frozen storage of surimi. In addition, the relative cryoprotective effects of whey protein concentrate, whey protein isolate, soy protein isolate, flaxseed meal and flaxseed protein were assessed in surimi during storage. / Raw surimi was prepared by repeatedly washing Alaska pollock flesh with chilled water. The product was either slowly frozen or underwent rapid freezing using liquid air; in either case it was then subjected to frozen storage at -20°C for 24 months. Protein structural changes were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, Fourier transform infrared/attenuated total reflectance (FTIR/ATR) spectroscopy, and differential scanning calorimetry (DSC). / FTIR/ATR spectroscopy showed that during preparation of surimi the alpha-helix content increased with increased number of washing cycles. DSC results revealed a shift in the thermal transition of actin to a higher temperature during surimi preparation. All electrophoresis, FTIR/ATR spectroscopy and DSC results revealed a loss of myofibrillar proteins from surimi after three washing cycles, suggesting that three washing cycles were adequate to prepare surimi. / Native-PAGE showed no major changes in surimi after 24 months storage at -20°C. SDS-PAGE showed relatively minor changes in protein subunit structure with some loss of the myosin light chains (MLC); myosin heavy chain (MHC), actin and tropomyosin were found to be relatively stable. FTIR/ATR spectroscopy indicated a significant decrease in alpha-helix relative to beta-sheet structure in surimi after 2 years of storage at -20°C. The loss of alpha-helical content was more significant in slowly frozen surimi compared to rapid-frozen surimi samples. DSC results revealed a shift in the thermal transition of actin to lower temperatures during frozen storage of surimi. / Changes in the ratio of alpha-helix to beta-sheet structures suggested that flaxseed protein was the most effective cryoprotectant, followed by whey protein isolate and soy protein isolate, for maintaining protein structure stability during frozen storage. Whey protein concentrate and flaxseed meal showed the least cryoprotective ability. After 15 days storage at 4°C, the SDS-PAGE results showed that flaxseed protein was the only cryoprotectant that prevented the degradation of myosin heavy chain, actin and myosin light chains.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

A casein agar diffusion method was developed to detect and quantify pertinent levels of proteinases produced in raw milk supplies by heat resistant Bacillus sporeformers. In order to optimize the required heat treatment conditions of raw milk samples, trials that involved a combination of different temperatures and times were evaluated. A heat treatment of 75°C for 20 min was the most effective for recovering the highest number of surviving spores. A sporulation broth containing five different minerals and supplemented with 0.2% nonfat dry milk was used to maximize spore production in all heat-treated samples. A β-casein based assay detected proteinase activity from raw milk samples that ranged from 0.093 to 4.034 units/mg which corresponded to zones of β-casein precipitation in the β-casein agar of 5.0 and 15.0 mm respectively, and was compared to Protease Type VIII (from B. licheniformis). This assay correlated well with the fluorescein isothiocyanate casein-labeled assay (FITC), R=0.995 (Protease Type VIII). Proteases of Bacillus origin such as Protease Type IX, X, XV and XXXI were also evaluated but were rejected in favor of a broader range of activity expressed by Protease Type VIII. For an initial set of 370 raw milk samples, no quality deterioration, such as coagulation or bitter taste was observed in heat-treated (75°C for 20 min) and incubated samples (7.2°C for 10 days). However, during the winter season, 18 of 75 incubated samples (7.2°C for 10 days) tasted slightly bitter and exhibited a slight degree of casein precipitation. One sample coagulated but exhibited no proteinase activity on the β-casein agar gel, hence it was considered a false negative. The positive results for proteinase activity from raw Grade A samples tested by the β-casein agar diffusion method did not correlate either with fresh spore counts (R=0.21) or post-heat treatment incubation counts (R=0.03) or with psychrotrophic sporeformer counts (R=0.06). The β-casein agar diffusion method is simple, rapid and sensitive to Bacillus spp. proteinases, but was unreliable in projecting results related to the psychrotrophic sporeformer count. Consequently, further research is required to establish optimum conditions (time and/or temperature) and inoculum volumes into sporulation broth for attainment of a more positive correlation between β-casein agar precipitation zones and psychrotrophic sporeformer populations of either raw or processed milk samples. / Graduation date: 1991

Guanidine-stable chymoelastase (GUSCE), a component of the protease mixture known as Pronase, has been shown to be stable and active under conditions which denature and inactivate most proteins. In the absence of denaturant, this enzyme has shown proteolytic specificity for phenylalanyl, tyrosyl, and leucyl peptide bonds. In the presence of denaturant, however, the cleavage specificity has not been defined.In order to determine the effects of denaturant on the cleavage specificity of GUSCE, six small peptides of known amino acid sequence were hydrolyzed by GUSCE in the presence and absence of 6.OM guanidinium chloride. The site of GUSCE cleavage was acertained by dansylation of the new N-terminal amino acids, which were produced by proteolysis, followed by thin layer chromatographic identification of the resulting dansylated amino acids.The results indicate that GUSCE catalyzed the hydrolysis of phenylalanyl and tyrosyl peptide bonds in the absence as well as the presence of 6.OM guanidinium chloride. Of the tyrosyl and phenylalanyl peptide bonds hydrolyzed, all were between non-terminal amino acids, which illistrates the endo-peptidase characteristics of GUSCE. With one exception, only those peptide bonds cleaved by GUSCE in the absence of denaturant were cleaved in the presence of denaturant. In the case of oxytocin, the presence of denaturant was actually required for the cleavage of the Tyr(2)-Ile(3) peptide bond. The demonstrated predictablilty of GUSCE cleavage in the presence of denaturant should greatly enhance its utility in the sitespecific proteolysis of insoluble or otherwise proteolysisresistant protein substrates.Ball State UniversityMuncie, IN 47306

Graduation date: 1993

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 181-216) Also available in print.