Chapter 14
Genetic Engineering of Bovine κ-Casein To Enhance Proteolysis by Chymosin
Downloaded by STANFORD UNIV GREEN LIBR on July 1, 2012 | http://pubs.acs.org Publication Date: February 19, 1991 | doi: 10.1021/bk-1991-0454.ch014
Sangsuk Oh and Tom Richardson Department of Food Science and Technology, University of California, Davis, CA 95616
k-Casein cDNA was mutated to change the chymosin sensitive site from a Phe(105)-Met(106) bond to a Phe(105)-Phe(106) bond which, in theory, should be attacked at a faster rate by acid proteases. Mutant k -casein and normal k-casein were expressed in E. coli strain AR68 using the secretion vector, pIN-III-ompA. The expressed k-caseins were extracted in urea buffer and partially purified using DE 52 anion exchange chromatography. Partially purified k-caseins were hydrolyzed with chymosin at 30°C. Initial hydrolysis rates were compared. The mutant k-casein (Phe(105) -Phe(106)) was hydrolyzed approximately 80 percent faster than the wild-type (Phe(105)-Met(106)) k-casein as determined using western blots, followed by immunochemical staining and laser gel scanning. The s t a b i l i t i e s o f p r o t e i n s t o e n z y m a t i c a t t a c k a r e t o a l a r g e e x t e n t d i c t a t e d by t h e s t r u c t u r e o f t h e amino a c i d s on e i t h e r s i d e o f t h e s u s c e p t i b l e bond and, i n some c a s e s , on t h e p r i m a r y sequence around the s c i s s i l e l i n k a g e . Moreover, exposure o f t h e bond t o e n z y m a t i c a t t a c k i s dependent upon i t s s t e r i c a v a i l a b i l i t y . Thus, t h e t h r e e d i m e n s i o n a l s t r u c t u r e o f a p r o t e i n may be i m p o r t a n t f o r e n z y m a t i c attack. The r a t e o f h y d r o l y s i s o f a g i v e n bond c a n have w i d e - r a n g i n g r a m i f i c a t i o n s on t h e f u n c t i o n a l c h a r a c t e r i s t i c s o f f o o d p r o t e i n s and on t h e q u a l i t y o f r e s u l t a n t f o o d p r o d u c t s . F o r example, i n t h e c o a g u l a t i o n o f m i l k t o make cheese, t h e Phe(105)-Met(106) bond o f kc a s e i n i s a t t a c k e d by chymosin ( r e n n i n ) . This r e s u l t s i n the loss of a p o l a r p e p t i d e from t h e C-terminus o f k - c a s e i n which d e s t a b i l i z e s t h e c a s e i n m i c e l l e s c o m p r i s e d o f
