Isolation and partial characterization of rennet-like proteases from Australian cardoon (Cynara cardunculus L.).
The yield, protein content, proteolytic activity, and substrate specificity of crude and partially purified extracts from dried and fresh Australian cardoon (Cynara cardunculus L.) flowers were determined. Crude water extracts had high yield but low protein content and proteolytic activity, whereas citric acid extracts had low yield but high protein content and proteolytic activity. Fresh flower extracts gave higher yield and proteolytic activity but lower protein content in comparison with dried flower extracts. Purification with ammonium sulfate resulted in significantly increased proteolytic activity for water extracts from both fresh and dried cardoon flowers, whereas the proteolytic activity of citric acid extracts did not change significantly after purification. Irrespective of extraction method, all extracts had higher proteolytic activity against ovine whole and kappa-caseins compared to their bovine counterparts, showing optimal activity at 37 degrees C and pH 6.0. Separation of purified extracts by ion-exchange liquid chromatography yielded three active fractions, each of which when assayed with sodium dodecyl sulfate capillary electrophoresis revealed two subunits with molecular masses of 15.5 and 33.1 kDa, respectively.
School of Wine and Food Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.
Shaojiang
S
Chen
Jian
J
Zhao
Samson
S
Agboola
D000645:Ammonium Sulfate; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D012085:Chymosin / Q000378:metabolism; D019343:Citric Acid; D031942:Cynara / Q000201:enzymology*; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D035264:Flowers / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D010936:Plant Extracts / Q000737:chemistry*; D011506:Proteins / Q000032:analysis; D013379:Substrate Specificity; D013696:Temperature; D014867:Water
D002364:Caseins; D010936:Plant Extracts; D011506:Proteins; D014867:Water; D019343:Citric Acid; C025350:rennet; D010450:Endopeptidases; D012085:Chymosin; D000645:Ammonium Sulfate