Research On Actinomucor Elegans Carboxypeptidase Based On RNA-Seq Technology And The Characteristics Of A Recombinant Carboxypeptidase (AecpY) Expressed In Pichia Pastoris

Actinomucor elegans,whichsecrete many kinds of proteases,is one of the majoy fungi that iscommonly used to produce bean curd,laba beans,black beans and other flavor food,Actinomucor elegans play an important role in the formation of bean curd flavor compounds in thepost-fermented process.Protease could catalyze hydrolysis of peptide bonds,there are more than 900 kinds of microbial protease which has been reported.The physical activity and occurrence of disease such as digestion and absorption,blood coagulation,hemolysis,inflammation,regulation of blood pressure,cell differentiation,aging organism,cancer metastasis,activation of the physiologically active peptide and so on are related with the protease.Serine protease is a kind of protease which active center is serine,and it is important in a wide range of biological organisms.Serine carboxypeptidase is a kind of eukaryotic protease,primarily exist in the plant or fungal vacuoles and animal lysosome,which belongs to ?/? hydrolase family and S10 family in the clan of SC carboxypeptidase.In this paper,we have analysed the transcriptome of Actinomucor elegansand found 7 kinds of carboxypeptidases,which are mainly in M28,S28 and S10 families.We also have analysed the gene sequences and evolutionary relationship about these carboxypeptidases.Than,we culture and observe the Actinomucor elegans on bran medium after 1-3 days.Clone and expression the Aecp Y gene of Actinomucor eleganscarboxypeptidase by construct the recombinant strain p ICh-Aecp Y-GS115.Purified the protein by desalination of enzyme liquid.The research revealed that the highest activity of r Aecp Y is at p H 5,suitable weakly acidic conditions.At 40 ?,the activity reached the highest and keeped stability during 20-50 ?.The inhibitor PMSF effect the activity of r Aecp Y greatly,but the inhibitor EDTA even don not effect the activity of r Aecp Y,hence it is supposed that r Aecp Y belongs to serine carboxypeptidase.In addition,the Zn²⁺ and Mn²⁺ could promote the enzyme activity,when Cu²⁺ and Fe²⁺ will inhibit.