Journal
CARBOHYDRATE POLYMERS
Volume 224, Issue -, Pages -Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.carbpol.2019.115135
Keywords
Chondroitin sulfate ABC lyase; Chondroitin sulfate; Protein evolution; Oligosaccharide; Secretory expression; Escherichia coli
Categories
Funding
- National Natural Science Foundation of China [31670092]
- National Key R&D Program of China [2018YFA0901401]
- Fundamental Research Funds for the Central Universities [JUSRP51707A]
- National first-class discipline program of Light Industry Technology and Engineering [1012050205190080]
- Program for Changjiang Scholars and Innovative Research Team in University [IRT_15R26]
- 111 Project
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Chondroitin sulfate ABC lyases (csABCs) have attracted intensive attention because of their wide potential applications in promoting tissue regeneration and generating oligosaccharides. In the present study, three csABC I encoding sequences were analyzed and site-directed mutagenesis results demonstrate that residues Leu125 and Leu322 are essential to activity and mutation of each leucine residue to proline dramatically decreased enzymatic activity. Additionally, our results showed that mutation of 1309 V significantly increased the catalytic efficiency. By recruiting OmpA signal peptide and engineering the permeability of cell membrane with deletion of a lipoprotein encoding gene lpp, all recombinant enzymes were secreted and the extracellular activity was finally increased to 2.99 +/- 0.1 U/mL in batch fermentation. More importantly, the engineered csABC I with high activity can rapidly degrade chondroitin sulfate to the end tetrasaccharides and disaccharides, demonstrating its applicability for preparation of chondroitin sulfate oligosaccharides.
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