TY - BOOK

T1 - New insights into the bovine Plasminogen system

AU - Nurup, Casper Normann

PY - 2021

Y1 - 2021

N2 - Plasminogen, the zymogen form of plasmin, is a serine protease that is transferred from blood to the milk and is responsible for the hydrolysis of casein in dairy products. Activity from plasmin can be detrimental or beneficial to an individual dairy product. Efficient control of plasmin activity is therefore required for the improvement of the functionality and quality of dairy products.The initial aim of this Ph.D. thesis was to develop an affinity chromatographic removal approach that could be used to remove plasmin activity from milk. It was believed that this could be used to obtain full control of plasmin activity in dairy products. However, during the work with the chromatographic removal ofplasmin activity from acid whey it was discovered that plasmin activity was divided into a bound-fraction, non-bound fraction, and lost activity fraction, which corresponded to 25, 50, and 25 % of the total plasmin activity, respectively. The non-bound fraction contained the lateral generated plasmin-fragments: midi-, mini-, and micro-plasmin. Further, the bound concentrated fraction contained a high molecular weight protein, that was larger than thatof full length plasmin. This high molecular weight protein exhibited plasmin like activity, and were indicated to be a complex or an association formed between plasmin and immunoglobulin. This indicates that the observed plasmin activity was more complex than expected. Due to this observed complexity, we decidedto study the different sized plasmin variants and their hydrolysis of casein in detail. Therefore, novel P. pastoris production protocols for the recombinant production of the different plasmin variants, full-length-, midi-, mini-, and micro-plasmin were developed. Difference in casein hydrolysis by the plasmin variants was studied using LC-MS/MS. Different marker peptides and their fragments from the hydrolysis of casein (aS1-, aS2-, and b-caseins), revealed that there was a difference in specificity, but also in the rate of the hydrolysis by the different plasmin variants.

AB - Plasminogen, the zymogen form of plasmin, is a serine protease that is transferred from blood to the milk and is responsible for the hydrolysis of casein in dairy products. Activity from plasmin can be detrimental or beneficial to an individual dairy product. Efficient control of plasmin activity is therefore required for the improvement of the functionality and quality of dairy products.The initial aim of this Ph.D. thesis was to develop an affinity chromatographic removal approach that could be used to remove plasmin activity from milk. It was believed that this could be used to obtain full control of plasmin activity in dairy products. However, during the work with the chromatographic removal ofplasmin activity from acid whey it was discovered that plasmin activity was divided into a bound-fraction, non-bound fraction, and lost activity fraction, which corresponded to 25, 50, and 25 % of the total plasmin activity, respectively. The non-bound fraction contained the lateral generated plasmin-fragments: midi-, mini-, and micro-plasmin. Further, the bound concentrated fraction contained a high molecular weight protein, that was larger than thatof full length plasmin. This high molecular weight protein exhibited plasmin like activity, and were indicated to be a complex or an association formed between plasmin and immunoglobulin. This indicates that the observed plasmin activity was more complex than expected. Due to this observed complexity, we decidedto study the different sized plasmin variants and their hydrolysis of casein in detail. Therefore, novel P. pastoris production protocols for the recombinant production of the different plasmin variants, full-length-, midi-, mini-, and micro-plasmin were developed. Difference in casein hydrolysis by the plasmin variants was studied using LC-MS/MS. Different marker peptides and their fragments from the hydrolysis of casein (aS1-, aS2-, and b-caseins), revealed that there was a difference in specificity, but also in the rate of the hydrolysis by the different plasmin variants.

M3 - Ph.D. thesis

BT - New insights into the bovine Plasminogen system

PB - Department of Food Science, Faculty of Science, University of Copenhagen

ER -