The bacterium Caulobacter crescentus secretes an adhesive polysaccharide called holdfast, which is the known strongest underwater adhesive in nature. The deacetylase encoded by hfs (holdfast synthesis) H gene is a key factor affecting the adhesion of holdfast. Its structure and function are not yet clear, and whether other polysaccharide deacetylases exist in C. crescentus is still unknown. The screening of both HfsH and its structural analogue as well as their purification from the artificial expression products of Escherichia coli is the first step to clarify these questions. Here, we determined the conserved domains of HfsH via sequence alignment among carbohydrate esterase family 4 enzymes and screened out its structural analogue (CC_2574) in C. crescentus. The recombinant HfsH and CC_2574 were effectively expressed in E. coli. Both of them were purified by chromatography from their corresponding productions in E. coli and were then functionally analyzed. The results indicated that a high deacetylase activity (61.8 U/mg) was observed in recombinant HfsH but not in CC_2574, which suggesting that HfsH might be the irreplaceable gene mediating adhesion of holdfast in C. crescentus. Moreover, the divalent metal ions Zn2+ , Mg2+ , and Mn2+ could promote the activity of recombinant HfsH at the concentration from 0.05 to 1 mM, but inhibit its activity when the concentration exceeds 1 mM. In sum, our study first realized the artificial production of polysaccharide deacetylase HfsH and its structural analogue, and further explored their functions, both of which laid the foundation for the development of new adhesive materials.
Keywords: Caulobacter crescentus; functional analogue; holdfast; polysaccharide deacetylase; soluble expression.
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