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Recombinant Chymosin

Recombinant Chymosin

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Published by Enzo Taping

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Published by: Enzo Taping on Jul 04, 2011
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12/15/2013

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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 .

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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 .

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

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

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

‡ 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.

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

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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 .

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