Elsevier

International Dairy Journal

Volume 15, Issue 11, November 2005, Pages 1130-1137
International Dairy Journal

Natural heterogeneity of chymosin and pepsin in extracts of bovine stomachs

https://doi.org/10.1016/j.idairyj.2004.10.003Get rights and content

Abstract

The two main components, chymosin and pepsin, in calf and adult bovine rennet show heterogeneity with respect to their charge and activity. A large number of single stomachs have been extracted and their isoenzymes have been analysed by HPLC. The results showed three genetic forms of chymosin A, B and C and these are present in individual stomachs in the following combinations: AA, BB, CC, AB, AC and BC. The C variant, which can be distinguished from the degradation product of chymosin A, has never been characterised before. All three chymosins have been purified and partially characterised. Also bovine pepsins show a natural variability: the ratio between the main pepsin fractions varies according to the age of the animal and it therefore provides a criterion to distinguish between pepsin from calf and adult bovine animals.

Introduction

Bovine stomachs and the commercial extracts thereof, calf rennet and adult bovine (ox) rennet, contain three types of aspartic proteases (EC 3.4.23): chymosin, pepsin and the minor component gastricsin (Szecsi, 1992). Chymosin is the dominating enzyme in calf rennet and pepsin is the main component in adult bovine rennet, whereas gastricsin is a minor component accounting for less than 1% of the total enzymes in calf rennet up to 5% in adult bovine rennet. Gastricsin has therefore not gained much attention.

The two main enzymatic components of calf/bovine rennet show heterogeneity in electrophoretical mobility due to differences in their amino acid sequence (chymosin) or different degree of phosphorylation (pepsins), both resulting in different net charge of each subcomponent (Foltmann, Pedersen, Kauffman, & Wybrandt, 1979; Martin, Trieu-Cuot, Corre, & Ribadeau-Dumas, 1982).

For bovine chymosin, three iso-enzymatic fractions A, B and C, mentioned in the order of decreasing milk-clotting activity, have been identified (Foltmann, 1966). Other authors (Asato & Rand (1972), Asato & Rand (1977) have found four forms of (pro)chymosin named A, B, C and D, separated by their electrophoretic mobility. These four components were not well characterised and it is doubtful if they represent different genetic forms. The two major chymosins A and B have been fully characterised including analysis of amino acid and gene sequences (Foltmann, Pedersen, Kauffman, & Wybrandt, 1979; Foltmann, 1992). It is generally agreed that chymosin A and B are allelic variants of a single gene locus (Donnelly, O’Callaghan, & Carroll, 1984), which differs by only one amino acid: the A form has an Asp in position 244 (according to the natural numbering of the chymosin sequence) where chymosin B has a Gly. There has been more doubt about chymosin C. Several papers (Foltmann, 1964; Foltmann, Pedersen, Jacobsen, Kauffman, & Wybrandt, 1977) conclude that chymosin C originates, at least partly, from autolytic degradation of chymosin A and Danley and Geoghegan (1988) describe the mechanism behind the formation of chymosin C.

Donnelly, Carroll, O’Callaghan, and Walls (1986) have, however proposed, based on the extraction of a few single stomachs, that a third allelic chymosin exists. The literature on chymosin C is not clear, but it has generally been believed that the C fraction obtained by chromatography of calf rennet is mainly degraded chymosin A.

Besides this, Harboe (1992) has proposed that further microheterogeneity, which has been observed in rennets and fermentation-produced chymosin (FPC), is due to easy deamidations of a specific asparagine number 160. This observation is supported by the finding of the code for an asparagine in the gene sequence but an aspartic acid in the protein sequence. Lately, Lilla, Caira, Ferranti, and Addeo (2001) have characterised the chymosin variants in rennets and other chymosin preparations by mass spectrometry. Chymosins A and B were each found in three active forms differing at the N-terminal end, one being three residues longer and the other being two residues shorter than the published chymosin, indicating that, at least under some conditions, the splitting site between the pro-part and the active chymosin can vary. Further, two degradation fragments were found in rennet: one corresponds to the degradation of chymosin A whereas the other seems new. However, neither the genetic variant chymosin C nor the deamidated forms of chymosin were detected in this last study.

No evidences for genetic variants of bovine pepsin have been found: its heterogeneity is due to an increasing degree of phosphorylation (0–3 phosphate groups/mol) in the different fractions (Martin, 1984). In total, five components have been reported (Martin, 1984; Martin, Trieu-Cuot, Corre, & Ribadeau-Dumas, 1982), each of which differs in some aspects of activity as well as in stability.

The relative amounts of these isoenzymes in commercial rennets have been observed to change in relation to different countries or between suppliers; it seems therefore of general interest to define the causes of their natural and/or technological (work in progress) variability. The aim of the present study is to investigate the natural variability of chymosin and pepsin isoenzymes in individual abomasal extracts and commercial rennets in order to improve the knowledge about the enzymatic composition of bovine rennets.

Section snippets

Preparation of stomach extract

Ninety frozen bovine abomasa were purchased from different countries and individually ground and extracted, activated and clarified by the following procedure. Mucosa, cut off from whole stomach, were mixed with equal amounts (by weight) of Milli-Q water and minced by blending for 1 min. The extract was centrifuged 15 min at 5000 g and the proenzymes in the supernatant were activated at pH 2.0 by sulphuric acid. After centrifugation for 15 min at 5000 g, pH in the supernatant was raised to 5.8 and

Results and discussion

In the HPLC ion-exchange chromatographic profile of a commercial calf rennet, as shown in Fig. 1, four isoenzyme fractions for chymosin and four–five fractions for pepsin can normally be distinguished. The comparison with the HPLC profiles of two fermentation-produced chymosin A and B allows identifying these two chymosin fractions in calf rennet (RT=18.6 and 17.4 min for A and B, respectively).

The two minor peaks eluted earlier (RT=15.9 and 15.0 min) could be the chymosin named C in the

Conclusions

A consistent and surprising variability has been observed in individual bovine stomachs, either in qualitative or quantitative distribution of chymosin and pepsin fractions. Three allelic forms A, B and C of chymosin exist and not only A and B as earlier believed. Their combinations follow a Mendelian model where six possible phenotypes were found. Two chymosin alleles are co-dominantly expressed in each single stomach. A degradation product of chymosin A, named A2, can be distinguished from

Acknowledgements

We thank Nadia Rossi, Instituto Sperimentale Lattiero Caseario, Lodi, Italy and Sari Charlotte Eltong, Chr. Hansen, Hoersholm, Denmark for very competent technical assistance.

References (21)

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