Purification of rennin

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35 . 6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bellshaped stability profile as a function of temperature with maximum activity near 55 8C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7 . 0. The ratio of milk clotting to proteolytic activity was 3 . 03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

Section: Resultssupporting

confidence: 65%

Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis)

Mohanty

Mukhopadhyay

Kaushik

et al. 2003

“…Crude chymosin was prepared by saturation of a commercial rennet solution (Hansen's standard rennet, Chr. Hansen's Laboratory Ltd, Reading, Berks, England) with NaCl as described by Castle & Wheelock (1971). This was further purified by the ehromatographic method of O' Leary & Fox (1974) and stored at 4 °C as a salt-free lyophilized powder.…”

Section: Methodsmentioning

confidence: 99%

Casein compositional studies: 1. The composition of casein from Friesian herd milks

Barry

Donnelly

1980

Casein composition in Friesian herd milks was examined by quantitative column chromatography on hydroxyapatite. Whole casein was resolved into 5 fractions: (1) y-casein + para-/e-casein; (2) K-casein + unidentified chymosin-resistant components; (3) unidentified minor protein; (4) /3-casein; (5) a sl 0 -+ minor a s -caseins. Protein in fractions 1, 2, 4, 5 was measured by ultraviolet spectrometry, using appropriate specific absorption coefficients. The quantitative contribution of chymosin-resistant components to the /e-casein fraction was measured by a method involving hydroxyapatite chromatography of chymosin-treated whole casein. Compositional analysis was carried out throughout a lactation cycle on a total of 24 casein samples from the milks of a winter/spring calving and autumn-calving herd. Average composition (expressed as % total casein), which was similar for both herds, was 48-7% a s -, 33-8% /?-, 4-9% y-, 9-5% K-and 3-2% unidentified components. Lactational effects on composition were also similar for both herds and were most pronounced in late lactation where increases were recorded in y-casein and unidentified components, and decreases in /?-and a s -caseins. Fluctuations in K-casein proportions were small and slightly higher values were obtained in early and late lactation. Casein composition also appeared to be affected to some extent by season, as shown by differences in the proportions of a s -caseins and unidentified components between mid-lactation milks from the 2 herds. Compositional variations resulted partly from the action of milk proteinase on /?-casein and also, apparently, from differences in relative rates of protein biosynthesis.

“…3.4.4.3) was prepared by salt fractionation and iso-electric precipitation of commercial rennet (Chr. Hansen, Reading, Berks) and purified by gel filtration (Castle & Wheelock, 1971).…”

Section: Methodsmentioning

confidence: 99%

Effect of varying enzyme concentration on the action of rennin on whole milk

Castle

Wheelock

1972

Self Cite

SummaryThe kinetics of the release of glycopeptides by the action of rennin on bovine whole milk have been studied. The initial rate of release of glycopeptides was proportional to the concentration of rennin. Using the integrated form of the Michaelis–Menten equation, values have been obtained forKmandV. These values varied between milk samples from individual cows, and for a single milk sample the value forKmincreased with increasing rennin concentration. It is suggested that theKmvalue is partly dependent on the carbohydrate composition of individual κ-casein molecules.