Camel and bovine chymosin: the relationship between their structures and cheese-making properties

Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105‐Met106 bond of the milk protein κ‐casein, releasing its predominantly negatively charged C‐terminus, which leads to the separation of the milk into curds and whey. Despite having...

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Published in:	Acta crystallographica. Section D, Biological crystallography. Vol. 69; no. 5; pp. 901 - 913
Main Authors:	Langholm Jensen, Jesper, Mølgaard, Anne, Navarro Poulsen, Jens-Christian, Harboe, Marianne Kirsten, Simonsen, Jens Bæk, Lorentzen, Andrea Maria, Hjernø, Karin, van den Brink, Johannes M., Qvist, Karsten Bruun, Larsen, Sine
Format:	Journal Article
Language:	English
Published:	5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01-05-2013 Wiley Subscription Services, Inc
Subjects:	Animals aspartic peptidases CALORIMETRY Camels Camelus Caseins - metabolism CATTLE Charging Cheese Chymosin Chymosin - chemistry Chymosin - metabolism CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY CRYSTAL STRUCTURE Crystallography, X-Ray CRYSTALS domain flexibility enzyme activity Enzymes FLEXIBILITY Glycosylation INTERACTIONS LOSSES Milk milk clotting Models, Molecular ORIENTATION Protein Conformation Research Papers RESOLUTION SHEETS STABILITY Static Electricity Structure-Activity Relationship substrate specificity SUBSTRATES surface charge SURFACES Thermal stability VARIATIONS aspartic peptidases surface charge domain flexibility milk clotting enzyme activity substrate specificity
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Summary:	Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105‐Met106 bond of the milk protein κ‐casein, releasing its predominantly negatively charged C‐terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel chymosin shows a 70% higher milk‐clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N‐terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin was redetermined at a slightly higher resolution (1.8 Å) than previously determined structures. Camel and bovine chymosin share the same overall fold, except for the antiparallel central β‐sheet that connects the N‐terminal and C‐terminal domains. In bovine chymosin the N‐terminus forms one of the strands which is lacking in camel chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids delineating the substrate‐binding cleft suggest a greater flexibility in the ability to accommodate the substrate in camel chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C‐terminus of κ‐casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk‐clotting activity of camel chymosin towards bovine milk.
Bibliography:	ark:/67375/WNG-63BLHFDR-1 istex:4CEBD3D272B7E2EB64513BC273CB5C5DFAE0FBBA ArticleID:AYDWD5199 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 Current address: Center for Prevention of Obesity, Diabetes and Cardiovascular Disease, Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA.
ISSN:	1399-0047 0907-4449 1399-0047
DOI:	10.1107/S0907444913003260