Abstract
Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.
Highlights
Chymosin, previously called rennin, is an aspartate proteinase which is found in the fourth stomach of suckling calves, among other animals (Ustunol and Hicks, 1990)
Submitted Jan. 13, 2016; Revised Feb. 14, 2016; Accepted Mar. 17, 2016. This signal sequence is removed to produce prochymosin. This zymogen is converted to active chymosin by cleavage of a 42-amino acid pro-peptide at the NH2terminus facilitated by the acidic conditions found in the stomach (Pedersen et al, 1979)
DNA technology and micro-organisms to produce recombinant rennet to fulfill the shortage of rennet
Summary
Previously called rennin, is an aspartate proteinase which is found in the fourth stomach of suckling calves, among other animals (Ustunol and Hicks, 1990) This enzyme can act against the Phe105-Met106 peptide bond which is present in milk k-casein molecules (Foltmann, 1969), generating insoluble para-k-casein and resultant clotting of milk (McMahon et al, 1984). Its critical importance in the production of cheeses combined with the current high demand for cheeses has resulted in a worldwide shortage of rennin This shortage is made more serious due to a contraction of the veal calf market; bovine rennet has traditionally been extracted from the abomasum of unweaned calves. In this paper we report the cloning, bioinformatic analysis, expression detection and enzymatic analysis of heterologous yak chymosin, and provide a novel chymosin for use in cheese production
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