Part:BBa_K4719011
AnCDA chitin deacetylase
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 348
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 361
Illegal XhoI site found at 593 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
Vilnius-Lithuania iGEM 2023 team's goal was to create a universal synthetic biology system for Komagataeibacter xylinus for in vivo bacterial cellulose polymer composition modification. Firstly, we chose to produce a cellulose-chitin polymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by K. xylinus.
Usage and biology
AnCDA is chitin deacetylase from Aspergilus nidulans FGSCA4. This deacetylase is a member of carbohydrate esterase family 4 (CE4) of the CAZy database ([http://www.cazy.org/, www.cazy.org]). It has broad substrate specifity – AnCDA hydrolyzes N-acetamido groups in chitin oligomers, crystalline chitin and chitosan, however, it does not deacetylase peptidoglycan. AnCDA reaches its highest level of activity at approximately 50°C and pH 8.0 when Co2+ is used as a cofactor.
AnCDA gene has three exons and encodes the primary product of 237 amino acids, including the N-terminal extracellular signal sequence. Coding regions except leader sequence were cloned into pBAD/HisB vector for recombinant protein expression in E. coli and protein purification using Ni-NTA chromatography with His-tag fused to AnCDA in N-terminal.
This basic part is used for achieving bacterial cellulose-chitosan polymer by enzymatic reaction of deacetylation from bacterial cellulose-chitin. In addition, we have created a construct BBa_K4719019 to improve the degree of deacetylation.
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