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Carboxypeptidase Y (E.C. 3.4.12.1) was produced from Kluyveromyces fragilis ATCC 28244. The maximum growth and enzyme production were obtained during 24 hr of growth at the late logarithmic phase with optimized conditions (25$ sp circ $C, 300 rpm, pH 5) using YPD (1% yeast extract, 2% peptone, 2% dextrose, w/v) broth medium. A Fast Protein Liquid Chromatography (FPLC) was used for the enzyme purification. The enzyme was purified to 216 fold over the crude extract with a recovery of 18%. The apparent molecular weight of the purified enzyme was estimated to be 120 kDa on Native-PAGE and 56 kDa on SDS-PAGE suggesting that carboxypeptidase Y from Kluyveromyces fragilis consists of two subunits. The pH and temperature optima of the enzyme were pH 6.0 and 35$ sp circ$C, respectively. The enzyme activity was strongly inhibited by diisopropylphosphofluoridate (DIPF) and phenylmethylsulfonylfluoride (PMSF), and caused an average 50% loss of activity when incubated with various metal cations. / The apparent K$ sb{ rm m}$ and V$ sb{ rm max}$ values obtained for n-benzoyl-L-tyrosine-p-nitroanilide (BTPNA) and carboxybenzoxyphenylalanylalanine (Cbz-Phe-Ala) were 5.1 mM and 13.4 $ mu$mole/min/mg and 2.98 mM and 22.58 $ mu$mole/min/mg, respectively. Carboxypeptidase Y hydrolysis on the tryptic digests of $ alpha sb{ rm s1}$- and $ beta$-casein showed that the enzyme randomly removed five and three hydrophobic peptides, respectively and greatly reduced the size and heights of the other peptides analysed on Reversed Phase-High Performance Liquid Chromatography (RP-HPLC).

A total of 853 milk samples with different phenotypes of $ kappa$-casein ($ kappa$-CN) and $ beta$-lactoglobulin ($ beta$-LG) and different preheating temperatures of 30, 70, 75 and 80$ sp circ$C were used for the making of individual laboratory scale Cheddar type cheese and for the determination of coagulating properties. Data obtained from milk input, cheese output and chemical analyses were used to calculate actual, 37% moisture adjusted and Van Slyke's theoretical yields and yield efficiency. Least squares analyses of data indicated that higher 37% moisture adjusted yields and yield efficiencies were obtained with milk types VII, VIII and IX, which have the B gene for $ kappa$-CN when compared to milk types I, II and III, which have the A gene for $ kappa$-CN irrespective of preheating temperatures. Moisture adjusted yield, 10.49%, was the highest when milk type VII containing $ kappa$-CN BB and $ beta$-LG AA phenotypes was preheated at 30$ sp circ$C, whereas milk type IX, which has phenotype BB for $ kappa$-CN and BB for $ beta$-LG, had the highest adjusted yields with values of 11.36, 11.91 and 11.99% when preheated at 70, 75 and 80$ sp circ$C, respectively. When cheese was made from milk preheated at 30$ sp circ$C, total solids (64.19%), fat (35.31%) and protein (25.82%) were highest in cheese obtained from milk types IX, VII and IX, respectively, all of which have the B gene for $ kappa$-CN. These three components (61.54%, 30.85% and 24.47%) were lowest in cheese made from milk types III, III and I, respectively, all of which have the A gene for $ kappa$-CN. Cheese with moisture content close to 39% were produced by milk types I and II preheated at 30$ sp circ$C. by milk types III, IV, V, VI, VII and VIII preheated at below 70$ sp circ$C and by milk type IX preheated at below 75$ sp circ$C. (Abstract shorted by UMI.)

A total of 596 milk samples with varying fat (3.0 to 4.0%) and protein (3.0 to 4.0%) contents were used to make laboratory-scale cheese and to determine coagulating properties. Higher levels of fat and protein in milk were associated with higher cheese yield. Milk protein has greater effect on cheese yield than milk fat. Adjusted yield increased by 1.91 and 1.29% for every percentage increase in the protein and fat of milk, respectively. Higher levels of fat in milk produced a cheese containing higher fat content and lower protein content. Similarly, higher levels of protein in milk produced higher protein content and lower fat content of cheese. Higher protein to fat ratio (or casein to fat ratio) in milk was associated with better efficiency of fat retention in the cheese. Casein retention in the cheese was not affected by the levels of fat and protein, or protein to fat ratio in milk. Milk adjusted for fat and protein resulted in delayed coagulation and a significant decrease in the curd firmness when compared with unadjusted bulk tank milk having the same levels of the two components.

To optimize conditions to prepare a good quality of enzyme-modified cheese (EMC), EMC samples were prepared by using combinations of Neutrase RTM/Lactobacillus casei enzymes, Neutrase RTM/Debitrase(TM), NeutraseRTM/Flavorzyme(TM) and NeutraseRTM/PalataseRTM. Based on the results obtained from sensory and RP-HPLC analysis, the optimal combinations to prepare a good quality of EMC were found to be: NeutraseRTM with (I) L. casei enzymes (aminopeptidase activity 86.4 LAPU/g and esterase activity 110.0 U/g), (II) Debitrase(TM) (aminopeptidase activity 22.0 LAPU/g), (III) Flavorzyme(TM) (aminopeptidase activity 6.5 LAPU/g), and (IV) PalataseRTM M (lipase activity 200 LU/g). / The water-soluble fractions of EMCs prepared with different enzyme combinations were subjected to RP-HPLC on a Delta Pack C18 column, and selected peaks were purified on the same column using a binary gradient. One peak from NeutraseRTM digest, five peaks from NeutraseRTM /Debitrase(TM) digest, and two peaks from NeutraseRTM /L. casei enzyme digest were purified and identified by API mass spectrometry. All the purified peptides contained active sites within their sequences. / The volatile compounds in a series of EMCs prepared by L. casei and commercial enzymes as well as Cheddar cheese (mild, old, extra old) were also identified by using Pyrolysis/GC/MS and dynamic headspace techniques. Overall, 5 ketones, 8 fatty acids, 3 alcohols and 2 aldehydes were detected in most of the samples using Py/GC/MS. Propanoic, hexanoic, octanoic, decanoic, dodecanoic and tetradecanoic acids were found to be the major fatty acids present in EMC prepared by L. casei enzymes. Dynamic headspace analysis revealed the presence of 17 compounds including fatty acids, ketones, alcohols, aldehydes, and hydrocarbon in most of the sample analyzed. The flavor of EMC seems to depend not on any particular key component, but rather on a critical balance of all components present.

Graduation date: 1992

Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [508]-524).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 143 p.; also includes graphics. Includes bibliographical references (p. 106-111). Available online via OhioLINK's ETD Center

Thesis (M.Inst.Agrar.)(Food Production)--University of Pretoria, 2006. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.