Application of MmPI in preparation of trypsin inhibitors
The present disclosure relates to the field of genetic engineering or enzyme engineering, and in particular to an application of MmBBK2 in preparation of trypsin and chymotrypsin inhibitors. The MmBBK2 has the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2. The present disclosure clarifies for the first time that MmBBK2 in mulberry leaves has both trypsin and chymotrypsin inhibitory activity, and reveals its physical and chemical properties. The MmBBK2 has good application prospects in preparing trypsin and chymotrypsin inhibitors. On the basis of knowing its physical and chemical properties, its activity may be accordingly eliminated, promoting the development and utilization of mulberry leaf resources in animal feed, providing new perspectives and ideas for the development and utilization of mulberry leaves in animal feed and health food, and improving economic benefits of mulberry resources.
1 . A method of preparing a trypsin and chymotrypsin inhibitor composition, comprising adding an effective amount of MmBBK2 protein, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, to a composition comprising a protease inhibitor; and
wherein SEQ ID NO: 1 is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 3, and SEQ ID NO: 2 is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 4.
2 . A plasmid, comprising:
the nucleotide sequence of SEQ ID NO: 3 inserted between the Nde I and Not I restriction sites; or
the nucleotide sequence of SEQ ID NO: 4 inserted between the Ase I and Not I restriction sites.
3 . A host strain transformed with the plasmid of claim 2 , wherein the host strain is Escherichia coli.
4 . A method of eliminating trypsin and chymotrypsin inhibitory activity of the MmBBK2 protein of claim 1 , comprising:
1) Subjecting the MmBBK2 protein to a pH of 3-4;
2) incubating the MmBBK2 protein at a temperature of 121° C. and a pressure of 0.21 MPa for 20 minutes;
3) incubating the MmBBK2 protein with β-mercaptoethanol at a temperature of 100° C. for 10 minutes; or
4) performing a Maillard reaction on the MmBBK2 protein in the presence of glucose.