A Thermolabile Aspartic Proteinase from Mucor mucedo DSM 809: Gene Identification, Cloning, and Functional Expression in Pichia pastoris
Küçük Resim Yok
Tarih
2013
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Humana Press Inc
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
In this study, the cDNA encoding the aspartic proteinase of Mucor mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZ alpha A and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 A degrees C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 A degrees C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.
Açıklama
Anahtar Kelimeler
RACE, Mucor mucedo, Pichia pastoris, Aspartic proteinase, Milk clotting enzyme, Expression
Kaynak
Molecular Biotechnology
WoS Q Değeri
Q2
Scopus Q Değeri
Cilt
54
Sayı
2
