Biochemical and Molecular Characteristics of a Novel Hyaluronic Acid Lyase from Citrobacter freundii

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Abstract
The Gram-negative strain of Citrobacter freundii, YNLX, has the ability to degrade hyaluronic acid. In this study, we expressed a C. freundii hyaluronic acid lyase, from polysaccharide lyase family 8, in Escherichia coli. The purified recombinant enzyme (rHynACF8) showed a substantially higher cleavage activity of hyaluronic acid than chondroitin sulfate. We found that its optimal pH and temperature are 5.5 and 35 °C, respectively. In addition, the enzyme activity was not notably affected by most metal ions. Km and kcat of rHynACF8 towards HA were 1.5 ± 0.01 mg/mL and 30.9 ± 0.5 /s, respectively. rHynACF8 is an endo-acting enzyme. Its cleavage products had dramatically increased antioxidant activity than hyaluronic acid in vitro (p < 0.001). As the molecular weight of hyaluronic acid decreased, the intramolecular interactions among antioxidant functional groups were removed; in the process of the cracking reaction, new double bonds formed and conjugated with the carbonyl group. We presumed that the structural change is the critical factor influencing antioxidant capacity. Overall, we found that rHynACF8 from Gram-negative bacteria with metal ion resistance, indicated the relationship between the function and structure of its antioxidant cleavage product.
Funding Information
  • National Key Research and Development Program of China (2017YFB0308401, 32060214, 2019B020218003, 202101AT070069, YNWR-QNBJ-2020-124, S202110681017)
  • National Natural Science Foundation of China (2017YFB0308401, 32060214, 2019B020218003, 202101AT070069, YNWR-QNBJ-2020-124, S202110681017)
  • Research and Development Projects in important areas of Guangdong Province (2017YFB0308401, 32060214, 2019B020218003, 202101AT070069, YNWR-QNBJ-2020-124, S202110681017)
  • Yunnan Fundamental Research Projects (2017YFB0308401, 32060214, 2019B020218003, 202101AT070069, YNWR-QNBJ-2020-124, S202110681017)
  • Yunnan Ten Thousand Talents Plan Young (2017YFB0308401, 32060214, 2019B020218003, 202101AT070069, YNWR-QNBJ-2020-124, S202110681017)
  • Yunnan Innovation and Entrepreneurship Training Program for College Students of China (2017YFB0308401, 32060214, 2019B020218003, 202101AT070069, YNWR-QNBJ-2020-124, S202110681017)

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