Difference between revisions of "Part:BBa K3195004"
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===Microcystin Degradation=== | ===Microcystin Degradation=== | ||
Similar to subtilisin, our subtilisin-like protease found in amphioxus (Branchiostoma)<sup>[1]</sup> can catalyze the hydrolysis of protein to amino acid. Subtilisin has been widely used in all walks of life even in laundry. | Similar to subtilisin, our subtilisin-like protease found in amphioxus (Branchiostoma)<sup>[1]</sup> 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. Then we test the activity of this enzyme and measured by HPLC-MS-MS. | + | 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 Then we test the activity of this enzyme and measured by HPLC-MS-MS. |
[[Image:T--SEU-Nanjing-China--SLPDegra.png|900px|thumb|center|<b>Figure 1.</b>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).<b>The sample 12(in red) was degraded by subtilisin-like protease.</b>]] | [[Image:T--SEU-Nanjing-China--SLPDegra.png|900px|thumb|center|<b>Figure 1.</b>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).<b>The sample 12(in red) was degraded by subtilisin-like protease.</b>]] | ||
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 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. |
Revision as of 03:15, 19 October 2019
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 Then we test the activity of this enzyme and measured by HPLC-MS-MS.
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 .
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 447
Illegal XhoI site found at 1645 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 889
Illegal AgeI site found at 1120 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 967