Prochymosin

Procedures for extraction of chymosin from veils were described by Ernstrom and Wong (1974). Crude rennet extract contains active chymosin and an inactive precursor (prochymosin). Addition of acid to the extract facilitates conversion of prochymosin to chymosin and allows the extract to reach maximum activity. Even though activation at lower pH is faster, poor stability of chymosin below pH 5.0 in the pres-... 

Activation of prochymosin involves the splitting of peptides from the N-terminal end of prochymosin with simultaneous reduction in molecular weight from about 36,000 to 31,000. The rate of conversion increases markedly with decreasing pH below 5.0 (Rand and Emstrom 1964). At pH 5.0, NaCl concentrations up to 2M increase the rate of activation. Milk-clotting activity plotted against activation time at pH 5.0 shows the course of activation (Fig. 12.1) to be autocatalytic. If activation is carried out in the presence of preformed chymosin, the S-shape disappears and the initial rate of the activation process increases with increasing concentration of preformed chymosin. Folt-... 

Harmsen MM, Bruyne MI, Raue HA et al (1996) Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant thaumatin in yeast. Appl Microbiol Biotechnol 46 365-370... 

Chymosin protease (1) Aspergillus niger var. awamori d-calf prochymosin gene (2) Escherichia coli K-12 d-calf prochymosin gene (3) Kluyveromyces marxianus cleaves a single bond in kappa casein cleaves a single bond in kappa casein 3.4.23.4... 

Chymosin (Aspergillus niger var. awamori, Escherichia coli K-12, and Kluyveromyces marxianus, each microorganism containing a calf prochymosin gene), 786, (S3)20 Chymotrypsin, 786, (S3)18 Chymotrypsin Activity, 793 Cinene, 518 1,8-Cineol, 496 Cineole, Percentage of, 818 Cinnamal, 468 Cinnamaldehyde, 468, 611 Cinnamic Acid, 468, 565, 612, (S3)66 Cinnamic Alcohol, 470 Cinnamic Aldehyde, 468 Cinnamon Bark Oil, Ceylon Type, 101, 578... 

Chymosin is secreted by the abomasum in the form of an intially inactive precursor, prochymosin, which is converted autocatalytically to chymosin (see below, p. 176). Many reviews on the properties of the zymogen and the enzyme have been published. One of the most recent, by Foltmann (J), also contains references to earlier reviews. The action of chymosin on K-casein is primarily responsible for the milk-clotting process. The numerous studies of this reaction have been reviewed recently by Mackinlay and Wake (32). Because of the number of diflFer-ent milk proteins and the complexity of their interactions, especially in the casein micelle, the full details of the coagulation process are not yet understood. 

Amino Acid Compositions. Amino acid compositions of mammalian pepsinogen and pepsin, gastricsin, and prochymosin and chymosin have been collected by Fruton (46), Those of five fungal enzymes have been collected by Matsubara and Feder (29). 

The size distribution and density of inclusion bodies have been studied only for two proteins, calf prochymosin and y-interferon (64). The mean particle sizes of y-interferon and prochymosin inclusion bodies were O.Slgm with a standard deviation of 0.17gm and 1.28gm with a standard deviation of 0.46gm, respectively. The buoyant density of the y-interferon and prochymosin inclusion bodies was proportional to the density of the suspension medium indicating a relatively high voidage within the particles. The void fraction (=void volume/total volume) was 70% for y-interferon and 85% for prochymosin. 

Kinetics of Inclusion Body Formation in Escherichia coli CY15070 Producing Prochymosin... 

The pWHA43 plasmid contains 3.9 kbp and replicates with the pMBl origin of replication. The plasmid was constructed with an insert of cDNA encoding met-prochymosin under the control of a tandem lac-trp promotor operator arrangement as described in (1). The prochymosin gene was inserted just upstream from the ampicillin resistance gene without a transcription terminator sequence between, but with the RNA polymerase binding site for amp also intact, so that presumably amp is transcribed from both the lac-trp and natural amp promotors. 

SDS gels were stained with Coomassie Blue and scanned using an LKB 2222-20 ultrascan XL densitometer. The amount of prochymosin was reported relative to the amount of a constitutively produced protein located just above the prochymosin band. The expression of this protein was determined to be proportional to the overall protein concentration in the cell sample and therefore acted as an internal standard for prochymosin content. 

Influence of Inoculum Density on the Prochymosin Content of the Culture. 

The densitometer measurements show that the fraction of cells containing visible inclusion bodies correlated to the (relative) amount of prochymosin in the culture as a whole (Figure 7). This correlation appeared to hold both within a single culture as it entered stationary phase and between different cultures with different inoculum sizes. The correlation was fairly linear until about 80% of the cells contained inclusion bodies then the amount of prochymosin sharply increased relative to the other protein band. This corresponded to early stationary phase when there was a sudden increase in prochymosin cortent and an appearance of multiple inclusion bodies within each cell. 

Figure 7. Relationship between the fraction of cells containing inclusions during exponential growth and the relative prochymosin content of the cells. Inoculum density 0.14 CFU/mL (a) inoculum density 140 CFU/mL ( ).

The fraction of cells which contained visible inclusion bodies during exponential growth was constant but depended on the inoculum size. Larger inocula yielded cultures with significantly fewer inclusion body containing cells. The amount of prochymosin correlated to the number of ceUs showing visible inclusions. 

The pole age model does not explain why low inoculum densities resulted in a higher fraction of the cells with visible inclusion bodies and higher synthesis rates for prochymosin without decreasing the exponential specific growth rate in the cells. 

Emtage JS, Angal S, Doel MT et al. (1983) Synthesis of calf prochymosin (prorennin) in Escherichia coli. Proc Natl Acad Sci USA 80(12) 3671-3675 Epstein W, Rothman-Denes LB, Hesse J (1975) Adenosine 3 5 -cycUc monophosphate as mediator of cataboUte repression in Escherichia coli. Proc Natl Acad Sd USA 72(6) 2300-2304 Erson B, Janson JC, Ryden L (1998) Introduction to protein purification. In Janson JC, Ryden L (eds). Protein purification principles, high-resolution methods, and applications, 2nd edn. Wiley-VCH, Weinheim, pp 3 0... 

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