Part:BBa_K3195004

Trx-His-EK tagged subtilisin-like protease

Subtilisin-like protease is a kind of alcalase found in amphioxus (Branchiostoma) similar to subtilisin protease. In our project, it has been used to degrade microcystin with another enzyme called carboxypeptidase Z/N.

Usage and Biology

Microcystin Degradation

Similar to subtilisin, our subtilisin-like protease found in amphioxus (Branchiostoma)^[1] can catalyze the hydrolysis of protein to amino acid. Subtilisin has been widely used in all walks of life even in laundry.

Interestingly, we found the subtilisin-like protease can degrade not only proteins but also microcystins.BBa_K3195004 was created for mass expression of this enzyme in E.coli(BL21). We remove the signal peptide and optimize its condons. Moreover, we added Trx-His-EK tag at N-terminals for the convenience of purification.We test the activity of this enzyme in degrading microcystin and measured by HPLC-MS-MS.

Figure 1.The abscissa represent 28 enzymes in our project and the unit of ordinate value is mg/L. The ordinate value represents the remained microcystin RR(MC-RR) in the degraded sample. The right picture shows the results of degradation by each enzyme, and the left one shows the results of mixing each enzyme with enzyme 8(carboxypeptidase Z/N).The sample 12(in red) was degraded by subtilisin-like protease.

In the wet lab experiments, we found that carboxypeptidase Z/N and subtilisin-like protein could not decompose microcystins independently, but when the two enzymes were put into the experiment, they could decompose one of the microcystins, microcystin RR. In our project, we cloned this part into pET32a vector and transformed it into E.coli(DH5α) for mass production of subtilisin-like protease .

In addtition for better understanding its mechanism we predicted its three-dimensional structure. By comparing its sequence with cathepsin B, we found several action sites of cathepsin B could find similar short peptide structures in these two amino acid sequences. CYX-184 was found in subtilisin-like protease.

Figure 1.The three dimension prediction of subtilisin-like protease.

We speculate that subtilisin-like protease are involved in the decomposite of microcystin RR, howvever due to some reasons in its degradation microenvironment, it cannot limit the movement(rotation,vibration,ect) of microcystin RR. Only together with carboxypeptidase Z/N, they can decompose microcystin RR.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 889
  Illegal AgeI site found at 1120
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 967