• Asian-Australasian Journal of Animal Sciences
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• v.29 no.9
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• pp.1363-1370
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• 2016

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 $pPICZ{\alpha}A$, 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.

• Journal of the East Asian Society of Dietary Life
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• v.7 no.3
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• pp.289-300
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• 1997

Newborn dog chymosin was extracted from the stomachs of dogs of 2 weeks of age, and was purified by ion exchange chromatography. Half of the sequence was determined by amino acid sequencing and the complete sequence was deduced from a cloned chymosin cDNA Results showed that the zymogen showed 79% sequence identity with calf prochymosin and 54% identity with porcine pepsinogen A The size of the propart and location of the residue which becomes the amino-terminus in the active enzyme was the same in the prochymosins. The maximum general proteolytic activity at pH 3.2 of newborn dog chymosin was 3-4% of that of porcine pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of newborn dog chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.2 of stomach extracts of individual dogs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or amino-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and newborn dog chymosin differ in the following positions (porcine pepsin numbering, subsites in parentheses) : Ser 12 Thr(S$_4$), Leu 30 Val(S$_1$/S$_3$), His 74 Gln(S'$_2$), Val 111 Ile(S$_1$/S$_3$), Lys 220 Met(S$_4$). With regard to the low general proteolytic activity of newborn dog chymosin, the substitution Asp303 Val relative to calf chymosin may contribute to an explanation of this.