Lorenzo L.

Taping III BS Biology

 Also known as rennin Main coagulating enzyme found in rennet which is used extensively in cheese production.

 hydrolyses a specific bond (Phe105 Met106) in kappacasein of milk kappa-casein acts as micelle stabilizer Precipitation of insoluble constituents

Cheese curd

Whey

 Calf abomasums as the traditional principal source

Abomasum Rumen

Reticulum

Omasum

 increasing demand shortage of calf stomachs ethical issues with animal slaughtering

Increase chymosin production has been made through expression of calf chymosin gene in recombinant K. lactis.

 Nontoxicogenic GRAS microorganism approved by US FDA Unlike P. pastoris, it does not require methanol to induce protein secretion Unlike E. coli, it doesn t secrete the expressed protein enclosed in inclusion bodies

Disruption of PMR1 in other yeast species (S. cerevisiae, P. pastoris, and H. polymorpha) increase the secretion of heterologous protein leading to the hypothesis that disrupting PMR1 in K. lactis would have the same effect.

 encodes for a P-type Ca2+-ATPase localized in the Golgi apparatus provides the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting and endoplasmic reticulumassociated degradation of tagged proteins

 Improper sorting of synthesized proteins secretion of partially folded proteins increase in the secretion of heterologous proteins normally destined for degradation in the lysosome.

Microbial Strains  K. lactis GG799 (type strain)  E. coli JM109 Media  Yeast Potato Dextrose for yeast  Luria-Bertani for bacterium

SalI pUC18-prochymosin

BlnI pKLAC1

SalI

BlnI

ligation Prochymosin gene

pKLAC1-N-prochymosin Transformed into E. coli JM109

SacII

SacII

Linearised cassette

Linearised cassette  transformants were selected using Yeast Carbon Base agar containing acetamide K. lactis GG799

Transformed K. lactis GG799

 Incubated in shake flasks containing YPD culture supernatants was added to 5mL of a suspension of non-fat milk powder and incubated until a clot formed One unit of chymosin activity was defined as the amount of active chymosin required to produce a clot in 40 min